James M. Paci, MD

ABSTRACT

In this study, we determine the in vivo effects of injecting sub-populations of leukocytes into normal rat Achilles tendons via a controlled laboratory study. Allogenic monocytes, granulocytes, or plasma were injected into 24 healthy rat Achilles tendons. Treated and contralateral un-treated control tendons then assessed for cellularity, histologic morphology, and vascularity after 7 and 14 days. Significant increases of 221% and 249% in cellularity (P = 0.014) were seen on day 14 within Achilles tendons injected with granulocytes as compared to plasma and monocytes, respectively. Also, significant improvement in morphology (P = 0.029) between days 7 and 14 was seen for the granulocyte injected Achilles tendons. Significant increases in cellularity after an injection of granulocytes, compared to monocytes and plasma, corresponds to a significant increase in inflammation within the tissue, suggesting that leukocyte-rich platelet-rich plasma (PRP) preparations are proinflammatory and potentially catabolic when injected into tendon tissue. The concentration and composition of white blood cells within PRP preparations is variable and needs to be better understood in order to optimize clinical utility of PRP injections.

Continue to: Tendinopathies are debilitating conditions...

Tendinopathies are debilitating conditions affecting patients worldwide every day. They arise most frequently from tendon overuse resulting in pathology.¹ There are 2 major subtypes of tendinopathy: tendinosis and tendinitis. Tendinosis, the more common condition, is characterized by long-term, chronic degradation of tendon tissue resulting in fibrosis from infiltrating fibroblasts.² Tendinitis, the less common condition, is characterized by an acute inflammatory response and inflammatory cell infiltrate.² Both conditions are common, with Achilles tendinopathy affecting 11% of runners and lateral epicondylitis affecting 1% to 3% of the general population.^3,4 Many sports-related overuse injuries, such as tendinopathies, go undiagnosed for extended periods of time because medical attention is avoided in order to prevent time loss from training or competing.⁵ These delays could be eliminated if a non-surgical option for treating tendon pathology was available.

Tendinopathies are believed to result from tendon overuse that causes micro-damage to collagen, as well as from significant changes in protein and enzyme composition within the tendon.⁶ The damage accumulates over time and eventually leads to chronic inflammation or fibrotic change within tendons, in both cases weakening the tendon and causing pain. Currently, accepted treatments for tendinopathies include: nonsteroidal anti-inflammatory drugs, physical therapy, ultrasound, laser-therapy, corticosteroids, glyceryl trinitrate patches, extracorporeal shock wave therapy, sclerotherapy, and surgery.⁷ Recently, platelet-rich plasma (PRP) therapy has emerged as a promising treatment for tendinopathies, as well as a variety of other orthopedic indications.

PRP consists of autologous blood from the patient, centrifuged to increase the amount of platelets in the sample above baseline, and subsequently injected around an affected tendon or joint.⁸ PRP is used to treat tendinopathy because it can supply injured tendons with blood components that aid in healing, which tendons do not receive due to poor vascularity.⁹ These components include growth factors, such as platelet derived growth factor (PDGF), transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF), endothelial growth factor, and leukocytes that can stimulate an inflammatory response within the injured tissue.¹⁰ The inflammatory response from the PRP induces a more robust reconstruction and revascularization of the injured tissue, stimulating proliferation, and remodeling.^11,12However, significant variability exists within the platelets, leukocytes, and growth factors that comprise PRP. This is attributed to 3 major causes. First, current commercial preparations of PRP result in differing platelet concentrations, as well as leukocyte-rich and leukocyte-poor compositions.^13,14 Variability in platelet concentrations results in unreliable amounts of growth factors, including cytokines, TGF-β, PDGF, VEGF and basic fibroblast growth factor in each preparation, while leukocyte levels affect inflammation, all leading to variable effects for each preparation.^15,16Second, despite sex and age of the PRP donor not being significant factors influencing variation in growth factor concentrations, the existence of an unexplained variation in concentrations of growth factors between different donors has been observed.¹⁷ Third, the selection of activating agents, bovine thrombin or calcium chloride, and their application, whether to the elbow, shoulder, or knee, produces variability.¹⁸

While the effects of platelets and growth factors in PRP have been well studied, less is known about the effects of differing cell types. Recently it was reported that the concentrations of leukocytes directly affect the outcomes of PRP injections. McCarrel and colleagues^19,20 found that as the number of leukocytes increased, there was a concomitant increase in the expression of inflammatory cytokines and catabolic activity. This effect may result in inferior healing of injured tissues and is attributed to the release of pro-inflammatory cytokines such as interleukin-1β from the leukocytes.²¹ There is also evidence that minimizing the catabolic effect of leukocytes may be just as important to tissue healing as the maximizing anabolic effect of platelets and growth factors.²²

The use of PRP has been highly disputed in recent years due to conflicting reports of its success in treating orthopedic conditions. Numerous favorable studies have shown benefit for treating chronic and acute orthopedic injuries including; rotator cuff tear repair, chronic refractory patellar tendinopathy, and chronic lateral tendinosis/epicondylitis.^23-26 Concurrently, articles demonstrating no significant effects from PRP have also been published. One study claiming that PRP injections did not improve outcomes of chronic Achilles tendinopathy did not differentiate whether patients had tendinosis or tendinitis, and did not consider leukocyte concentration in their PRP preparations²⁷ Another study that determined PRP is not beneficial to the healing of ruptured Achilles tendons after surgical repair also failed to consider the concentration of leukocytes in their PRP preparations.²⁸ One of the difficulties in comparing these studies is their heterogeneous nature. This arises from the use of different conditions in each study that makes the studies incomparable. Variations in PRP preparations lead to different concentrations of growth factors, platelets, and leukocyte concentrations. Additionally, tendinopathy models were not specified as tendinosis and tendonitis, and models or patients were not controlled for age, sex, or comorbidities. Given that leukocyte-rich and leukocyte-poor PRP preparations are currently widely used in clinical practice, the discovery of which type of preparation is indicated in which setting is paramount to evidence-based use of this treatment modality. Due to reports suggesting that leukocytes may be detrimental to tendon healing, determining which types of leukocytes are responsible for these effects is vital. As such, the purpose of this study is to determine the in vivo effects of sub-populations of leukocytes on normal rat tendons. This study design allowed us to isolate the effects of the injections to induce a response and remove confounding effects of normal healing response to a damaged tendon and effects from the injection itself. Our hypothesis was that the injection of leukocytes would cause an inflammatory response in rat tendons, leading to catabolic outcomes.

Continue to: METHODS...

 

 

METHODS

This was a prospective, in vivo, placebo controlled, randomized animal study. The University’s Institutional Animal Care and Use Committee approved all procedures prior to initiation. Twenty-four male Sprague-Dawley rats were randomized to 3 treatment groups (n = 8): monocytes; granulocytes, and; plasma, as a negative control.

Allogenic blood from 6 additional rats was collected into K2EDTA tubes via cardiac puncture. Allogenic, as opposed to autogenic, blood is commonly used in rat models because of low immunogenic response to blood from rats of the same strain and litter.^29,30 The blood was then pooled and the red cells lysed by incubation with Red Blood Cell Lysis Buffer (Roche). The samples were then sorted into fractions containing monocytes and granulocytes using fluorescence activated cell sorting (FACS) using a FACSAria (BD Biosciences). Cells were sorted using Purified PE Mouse Anti-Rat CD11b/c antibodies (BD Pharmingen) specific to monocytes, granulocytes, macrophages, dendritic cells, and microglia, APC-Cy7 Mouse Anti-Rat CD45 antibodies (BD Pharmingen) specific to all hematopoietic cells except erythrocytes, and FITC Mouse Anti-Rat CD42d antibodies (BD Pharmingen) specific to megakaryocytes and platelets. 20 μL of 0.2 mg/mL CD11b/c, 20 μL of 0.2 mg/mL CD 45, and 10 μL of 0.5 mg/mL CD42d antibodies were added to 1 mL of condensed non-red cells collected from the 6 rats and incubated at room temperature in the dark for 15 minutes. A fraction containing only platelet-poor plasma was also collected. For all treatments the injection volume was 75 μL. Rats in the monocyte group were injected with 200,000 cells in platelet-poor plasma, those in the granulocyte group were injected with 900,000 cells in platelet-poor plasma, and rats in the plasma control group received only platelet-poor plasma. The cell concentrations were based on previous studies that documented these concentrations that are found in typical leukocyte-rich PRP preparations.¹³

The animals were anesthetized with isoflurane gas and then injected aseptically once into their right Achilles tendon. The left Achilles tendon was used as an un-injected control, giving a total of 48 total Achilles tendons studied. At days 7 and 14 post-injection, 4 rats from each group were euthanized and the Achilles tendons were harvested.

The tendons were fixed in neutral buffered formalin for 24 hours and then embedded in paraffin and sectioned sagittally at 12 μm. The tendons were then stained with hematoxylin and eosin (H&E) using standard histological protocols and examined by 3 individuals trained to assess cellularity and morphology. All samples were assigned unrecognizable numbers and randomized prior to examination by individuals. Cell counts were based on the number of nuclei present in 3 mid-tendon high-power fields (400x) per sample. Morphology was graded on a scale of 1 to 3, with 1 being a normal tendon and 3 having severe pathology with total loss of alignment and crimping on 3 low-power fields (100x) per sample (Figures 1A-1G).

Vascularity was assessed by immunohistochemical staining using Rabbit Polyclonal Anti-CD31 antibodies (Abcam), a marker for vascular endothelial cells, using a Vectastain ABC Kit (Vector Laboratories) system and the ImmPACT AEC Peroxidase (HRP) Substrate (Vector Laboratories). Following staining, automated image analysis was performed (Bioquant). Briefly, all areas that did not contain tendon were masked. CD31 positive areas were then quantified using global thresholding. Vascularity was then calculated as ratio of CD31 positive area to total tendon area. Analyses were performed on 3 mid-tendon medium-power (200x) fields per sample.

For cellularity and morphology, the results for the injected tendons were normalized to those of their contralateral untreated controls and reported as a percentage. Results for vascularity were compared directly between treated tendons. Differences were assessed between groups at each time-point using Independent Samples Median Tests. When significant differences were identified, pairwise comparisons were performed to identify the source of the differences. All analyses were conducted using SPSS (V22, SAS Institute) with significant differences determined for values of P < 0.05.

RESULTS

No significant differences in cellularity between groups were seen at day 7 (P = 0.368) (Figures 1A-1G). However, a significant difference in cellularity between groups was seen at day 14 (P = 0.014). Pairwise tests showed there to be a significant increase in the number of cells in the tendons treated with granulocytes from 221% and 249% in cellularity (P = 0.014) on day 14, as compared to both monocytes and plasma, respectively. Morphologically, no significant differences were seen between groups at either time-point (P = 0.091 for day 7 and P = 1.000 for day 14) (Figures 2A-2G). However, a significant improvement in morphology was observed from day 7 to day 14 in the granulocyte group from 60% to 165% (P = 0.029). Finally, no differences were seen in vascularity between treatment groups at either time-point (P = 0.368 for day 7 and P = 0.535 for day 14) (Figures 3A-3G).

Continue to: DISCUSSION...

 

 

DISCUSSION

Our hypothesis that the injection of leukocytes would cause an inflammatory response in rat tendons leading to catabolic outcomes was confirmed in the granulocyte group. It should be noted that prior to the catabolic outcome, there was a transient anabolic effect in the granulocyte group during the second week. Deterioration in morphology was observed in the tendons injected with granulocytes on day 7, which subsequently recovered in the following week. We found that injecting granulocytes into normal tendons resulted in an increase in inflammatory cellularity, when compared to monocytes and plasma injections. 

Limitations inherent in this study are those similar to other in vivo studies. To begin with, the results of injections into rat tendons may not be translatable to human tendons. Despite this limitation, the rat is a common model for tendon research.³¹ Another limitation is that this study injected healthy Achilles tendons, rather than tendons with preexisting tendinopathy. In a naturally occurring tendinopathy, there may be other factors present that interact with PRP, and this model negates the contribution of these factors. Finally, while the immunohistochemistry (IHC) and morphological data are clear, the cellularity data are not clear in identifying the type of cells that were increased by granulocyte injection.  However, the cells appeared rounded, resembling inflammatory infiltrate; a common cell type seen in tendons.² While fibroblasts are also a common infiltrate during chronic tendinopathy, they are generally flat and appear on H&E as long spindle shaped cells. Thus, we believe the increased cellularity of the tendons after granulocyte injections is representative of an increase in inflammation. The increased cellularity could be due to the increased number of cells injected into the tendon; however, our conclusions are consistent with the increased inflammation previously reported linking leukocytes to tendon inflammation.^20,22,32

In terms of morphology, we hypothesized that degenerative changes would be seen in the tendons that were injected with granulocytes due to the inflammatory action of these cells. As part of the granulocyte response, neutrophils release proteases and macrophages can stimulate collagen synthesis via fibroblasts, both causing change within the extracellular matrix.^33,34 Indeed, we observed a significant change in tissue morphology in the granulocyte group over the course of 14 days. As the degenerative and regenerative effects of granulocytes take time to present, this is likely what we observed to occur between day 7 and 14 after treatment. These observations are also consistent with prior observations that leukocyte-rich PRP injections can be detrimental to tendon healing, but beneficial to tissue degeneration in the setting of chronic tendonitis.²⁰

We hypothesized that the vascularity of the tendons would be similar in all preparations. This was based on previous studies demonstrating that the lack of platelets in the platelet-poor plasma fraction is sufficient to deplete VEGF, the angiogenic agent in PRP.³⁵ In this study, there were no observable differences in vascularity of platelet-poor plasma, monocyte, and granulocyte injections. We attribute this to the lack of VEGF in any of these preparations. The aforementioned study also showed that the lack of platelets in injection was enough to prevent the angiogenic effect of this treatment.³⁵

Continue to: The goal of this study was...

 

 

The goal of this study was to assess the morphology, cellularity, and vascularity of normal tendons after injections of different leukocyte populations. This is clinically important because of the potential to tailor future PRP injections on a patient-by-patient basis. In patients requiring an anabolic response, leukocyte-poor PRP may be the best option. In contrast, when patient pathology requires an inflammatory response to improve healing³⁶ or breakdown fibrotic tissue, as seen in tendinosis, leukocyte-rich PRP may be warranted. Further, properly controlled clinical studies are needed to validate these recommendations.

Limitations inherent in this study are those similar to other in vivo studies. First, the results of injections into rat tendons may not be translatable to human tendons. Despite this limitation, the rat is a common model for tendon research.³¹ A second limitation is that this study injected healthy Achilles tendons, rather than tendons with preexisting tendinopathy. In a naturally occurring tendinopathy, there may be other factors present that interact with PRP, and this model negates the contribution of these factors. Finally, while the IHC and morphological data show clear changes, the cellularity data are not clear in identifying the type of cells that were increased by granulocyte injection. However, the cells appeared rounded, resembling inflammatory infiltrate; a common cell type seen in tendons.² While fibroblasts are also a common infiltrate during chronic tendinopathy, they are generally flat and appear on H&E as long spindle shaped cells. The last limitation of this study is the lack of functional mechanical testing since, clinically, healing of the tendon is also related to the strength of the tendon. Thus, we believe the increased cellularity of the tendons after granulocyte injections is representative of an increase in inflammation. Moreover, our results are consistent with the increased inflammation previously reported linking leukocytes to tendon inflammation.^20,22,32 It is interesting to note that the increase in inflammation does not lead to an increase in vascularity as could be expected.

CONCLUSION

We found that the injection of leukocytes into healthy rat Achilles tendons increases inflammation, as evidenced by increased cellularity and disrupted morphology, which suggests that leukocyte-rich PRP preparations may be contraindicated in settings of acute tendonitis. However, these preparations may be useful for a specific subset of tendinopathies, including chronic tendinosis.

ABSTRACT

In this study, we determine the in vivo effects of injecting sub-populations of leukocytes into normal rat Achilles tendons via a controlled laboratory study. Allogenic monocytes, granulocytes, or plasma were injected into 24 healthy rat Achilles tendons. Treated and contralateral un-treated control tendons then assessed for cellularity, histologic morphology, and vascularity after 7 and 14 days. Significant increases of 221% and 249% in cellularity (P = 0.014) were seen on day 14 within Achilles tendons injected with granulocytes as compared to plasma and monocytes, respectively. Also, significant improvement in morphology (P = 0.029) between days 7 and 14 was seen for the granulocyte injected Achilles tendons. Significant increases in cellularity after an injection of granulocytes, compared to monocytes and plasma, corresponds to a significant increase in inflammation within the tissue, suggesting that leukocyte-rich platelet-rich plasma (PRP) preparations are proinflammatory and potentially catabolic when injected into tendon tissue. The concentration and composition of white blood cells within PRP preparations is variable and needs to be better understood in order to optimize clinical utility of PRP injections.

Continue to: Tendinopathies are debilitating conditions...

Tendinopathies are debilitating conditions affecting patients worldwide every day. They arise most frequently from tendon overuse resulting in pathology.¹ There are 2 major subtypes of tendinopathy: tendinosis and tendinitis. Tendinosis, the more common condition, is characterized by long-term, chronic degradation of tendon tissue resulting in fibrosis from infiltrating fibroblasts.² Tendinitis, the less common condition, is characterized by an acute inflammatory response and inflammatory cell infiltrate.² Both conditions are common, with Achilles tendinopathy affecting 11% of runners and lateral epicondylitis affecting 1% to 3% of the general population.^3,4 Many sports-related overuse injuries, such as tendinopathies, go undiagnosed for extended periods of time because medical attention is avoided in order to prevent time loss from training or competing.⁵ These delays could be eliminated if a non-surgical option for treating tendon pathology was available.

Tendinopathies are believed to result from tendon overuse that causes micro-damage to collagen, as well as from significant changes in protein and enzyme composition within the tendon.⁶ The damage accumulates over time and eventually leads to chronic inflammation or fibrotic change within tendons, in both cases weakening the tendon and causing pain. Currently, accepted treatments for tendinopathies include: nonsteroidal anti-inflammatory drugs, physical therapy, ultrasound, laser-therapy, corticosteroids, glyceryl trinitrate patches, extracorporeal shock wave therapy, sclerotherapy, and surgery.⁷ Recently, platelet-rich plasma (PRP) therapy has emerged as a promising treatment for tendinopathies, as well as a variety of other orthopedic indications.

PRP consists of autologous blood from the patient, centrifuged to increase the amount of platelets in the sample above baseline, and subsequently injected around an affected tendon or joint.⁸ PRP is used to treat tendinopathy because it can supply injured tendons with blood components that aid in healing, which tendons do not receive due to poor vascularity.⁹ These components include growth factors, such as platelet derived growth factor (PDGF), transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF), endothelial growth factor, and leukocytes that can stimulate an inflammatory response within the injured tissue.¹⁰ The inflammatory response from the PRP induces a more robust reconstruction and revascularization of the injured tissue, stimulating proliferation, and remodeling.^11,12However, significant variability exists within the platelets, leukocytes, and growth factors that comprise PRP. This is attributed to 3 major causes. First, current commercial preparations of PRP result in differing platelet concentrations, as well as leukocyte-rich and leukocyte-poor compositions.^13,14 Variability in platelet concentrations results in unreliable amounts of growth factors, including cytokines, TGF-β, PDGF, VEGF and basic fibroblast growth factor in each preparation, while leukocyte levels affect inflammation, all leading to variable effects for each preparation.^15,16Second, despite sex and age of the PRP donor not being significant factors influencing variation in growth factor concentrations, the existence of an unexplained variation in concentrations of growth factors between different donors has been observed.¹⁷ Third, the selection of activating agents, bovine thrombin or calcium chloride, and their application, whether to the elbow, shoulder, or knee, produces variability.¹⁸

While the effects of platelets and growth factors in PRP have been well studied, less is known about the effects of differing cell types. Recently it was reported that the concentrations of leukocytes directly affect the outcomes of PRP injections. McCarrel and colleagues^19,20 found that as the number of leukocytes increased, there was a concomitant increase in the expression of inflammatory cytokines and catabolic activity. This effect may result in inferior healing of injured tissues and is attributed to the release of pro-inflammatory cytokines such as interleukin-1β from the leukocytes.²¹ There is also evidence that minimizing the catabolic effect of leukocytes may be just as important to tissue healing as the maximizing anabolic effect of platelets and growth factors.²²

The use of PRP has been highly disputed in recent years due to conflicting reports of its success in treating orthopedic conditions. Numerous favorable studies have shown benefit for treating chronic and acute orthopedic injuries including; rotator cuff tear repair, chronic refractory patellar tendinopathy, and chronic lateral tendinosis/epicondylitis.^23-26 Concurrently, articles demonstrating no significant effects from PRP have also been published. One study claiming that PRP injections did not improve outcomes of chronic Achilles tendinopathy did not differentiate whether patients had tendinosis or tendinitis, and did not consider leukocyte concentration in their PRP preparations²⁷ Another study that determined PRP is not beneficial to the healing of ruptured Achilles tendons after surgical repair also failed to consider the concentration of leukocytes in their PRP preparations.²⁸ One of the difficulties in comparing these studies is their heterogeneous nature. This arises from the use of different conditions in each study that makes the studies incomparable. Variations in PRP preparations lead to different concentrations of growth factors, platelets, and leukocyte concentrations. Additionally, tendinopathy models were not specified as tendinosis and tendonitis, and models or patients were not controlled for age, sex, or comorbidities. Given that leukocyte-rich and leukocyte-poor PRP preparations are currently widely used in clinical practice, the discovery of which type of preparation is indicated in which setting is paramount to evidence-based use of this treatment modality. Due to reports suggesting that leukocytes may be detrimental to tendon healing, determining which types of leukocytes are responsible for these effects is vital. As such, the purpose of this study is to determine the in vivo effects of sub-populations of leukocytes on normal rat tendons. This study design allowed us to isolate the effects of the injections to induce a response and remove confounding effects of normal healing response to a damaged tendon and effects from the injection itself. Our hypothesis was that the injection of leukocytes would cause an inflammatory response in rat tendons, leading to catabolic outcomes.

Continue to: METHODS...

 

 

METHODS

This was a prospective, in vivo, placebo controlled, randomized animal study. The University’s Institutional Animal Care and Use Committee approved all procedures prior to initiation. Twenty-four male Sprague-Dawley rats were randomized to 3 treatment groups (n = 8): monocytes; granulocytes, and; plasma, as a negative control.

Allogenic blood from 6 additional rats was collected into K2EDTA tubes via cardiac puncture. Allogenic, as opposed to autogenic, blood is commonly used in rat models because of low immunogenic response to blood from rats of the same strain and litter.^29,30 The blood was then pooled and the red cells lysed by incubation with Red Blood Cell Lysis Buffer (Roche). The samples were then sorted into fractions containing monocytes and granulocytes using fluorescence activated cell sorting (FACS) using a FACSAria (BD Biosciences). Cells were sorted using Purified PE Mouse Anti-Rat CD11b/c antibodies (BD Pharmingen) specific to monocytes, granulocytes, macrophages, dendritic cells, and microglia, APC-Cy7 Mouse Anti-Rat CD45 antibodies (BD Pharmingen) specific to all hematopoietic cells except erythrocytes, and FITC Mouse Anti-Rat CD42d antibodies (BD Pharmingen) specific to megakaryocytes and platelets. 20 μL of 0.2 mg/mL CD11b/c, 20 μL of 0.2 mg/mL CD 45, and 10 μL of 0.5 mg/mL CD42d antibodies were added to 1 mL of condensed non-red cells collected from the 6 rats and incubated at room temperature in the dark for 15 minutes. A fraction containing only platelet-poor plasma was also collected. For all treatments the injection volume was 75 μL. Rats in the monocyte group were injected with 200,000 cells in platelet-poor plasma, those in the granulocyte group were injected with 900,000 cells in platelet-poor plasma, and rats in the plasma control group received only platelet-poor plasma. The cell concentrations were based on previous studies that documented these concentrations that are found in typical leukocyte-rich PRP preparations.¹³

The animals were anesthetized with isoflurane gas and then injected aseptically once into their right Achilles tendon. The left Achilles tendon was used as an un-injected control, giving a total of 48 total Achilles tendons studied. At days 7 and 14 post-injection, 4 rats from each group were euthanized and the Achilles tendons were harvested.

The tendons were fixed in neutral buffered formalin for 24 hours and then embedded in paraffin and sectioned sagittally at 12 μm. The tendons were then stained with hematoxylin and eosin (H&E) using standard histological protocols and examined by 3 individuals trained to assess cellularity and morphology. All samples were assigned unrecognizable numbers and randomized prior to examination by individuals. Cell counts were based on the number of nuclei present in 3 mid-tendon high-power fields (400x) per sample. Morphology was graded on a scale of 1 to 3, with 1 being a normal tendon and 3 having severe pathology with total loss of alignment and crimping on 3 low-power fields (100x) per sample (Figures 1A-1G).

Vascularity was assessed by immunohistochemical staining using Rabbit Polyclonal Anti-CD31 antibodies (Abcam), a marker for vascular endothelial cells, using a Vectastain ABC Kit (Vector Laboratories) system and the ImmPACT AEC Peroxidase (HRP) Substrate (Vector Laboratories). Following staining, automated image analysis was performed (Bioquant). Briefly, all areas that did not contain tendon were masked. CD31 positive areas were then quantified using global thresholding. Vascularity was then calculated as ratio of CD31 positive area to total tendon area. Analyses were performed on 3 mid-tendon medium-power (200x) fields per sample.

For cellularity and morphology, the results for the injected tendons were normalized to those of their contralateral untreated controls and reported as a percentage. Results for vascularity were compared directly between treated tendons. Differences were assessed between groups at each time-point using Independent Samples Median Tests. When significant differences were identified, pairwise comparisons were performed to identify the source of the differences. All analyses were conducted using SPSS (V22, SAS Institute) with significant differences determined for values of P < 0.05.

RESULTS

No significant differences in cellularity between groups were seen at day 7 (P = 0.368) (Figures 1A-1G). However, a significant difference in cellularity between groups was seen at day 14 (P = 0.014). Pairwise tests showed there to be a significant increase in the number of cells in the tendons treated with granulocytes from 221% and 249% in cellularity (P = 0.014) on day 14, as compared to both monocytes and plasma, respectively. Morphologically, no significant differences were seen between groups at either time-point (P = 0.091 for day 7 and P = 1.000 for day 14) (Figures 2A-2G). However, a significant improvement in morphology was observed from day 7 to day 14 in the granulocyte group from 60% to 165% (P = 0.029). Finally, no differences were seen in vascularity between treatment groups at either time-point (P = 0.368 for day 7 and P = 0.535 for day 14) (Figures 3A-3G).

Continue to: DISCUSSION...

 

 

DISCUSSION

Our hypothesis that the injection of leukocytes would cause an inflammatory response in rat tendons leading to catabolic outcomes was confirmed in the granulocyte group. It should be noted that prior to the catabolic outcome, there was a transient anabolic effect in the granulocyte group during the second week. Deterioration in morphology was observed in the tendons injected with granulocytes on day 7, which subsequently recovered in the following week. We found that injecting granulocytes into normal tendons resulted in an increase in inflammatory cellularity, when compared to monocytes and plasma injections. 

Limitations inherent in this study are those similar to other in vivo studies. To begin with, the results of injections into rat tendons may not be translatable to human tendons. Despite this limitation, the rat is a common model for tendon research.³¹ Another limitation is that this study injected healthy Achilles tendons, rather than tendons with preexisting tendinopathy. In a naturally occurring tendinopathy, there may be other factors present that interact with PRP, and this model negates the contribution of these factors. Finally, while the immunohistochemistry (IHC) and morphological data are clear, the cellularity data are not clear in identifying the type of cells that were increased by granulocyte injection.  However, the cells appeared rounded, resembling inflammatory infiltrate; a common cell type seen in tendons.² While fibroblasts are also a common infiltrate during chronic tendinopathy, they are generally flat and appear on H&E as long spindle shaped cells. Thus, we believe the increased cellularity of the tendons after granulocyte injections is representative of an increase in inflammation. The increased cellularity could be due to the increased number of cells injected into the tendon; however, our conclusions are consistent with the increased inflammation previously reported linking leukocytes to tendon inflammation.^20,22,32

In terms of morphology, we hypothesized that degenerative changes would be seen in the tendons that were injected with granulocytes due to the inflammatory action of these cells. As part of the granulocyte response, neutrophils release proteases and macrophages can stimulate collagen synthesis via fibroblasts, both causing change within the extracellular matrix.^33,34 Indeed, we observed a significant change in tissue morphology in the granulocyte group over the course of 14 days. As the degenerative and regenerative effects of granulocytes take time to present, this is likely what we observed to occur between day 7 and 14 after treatment. These observations are also consistent with prior observations that leukocyte-rich PRP injections can be detrimental to tendon healing, but beneficial to tissue degeneration in the setting of chronic tendonitis.²⁰

We hypothesized that the vascularity of the tendons would be similar in all preparations. This was based on previous studies demonstrating that the lack of platelets in the platelet-poor plasma fraction is sufficient to deplete VEGF, the angiogenic agent in PRP.³⁵ In this study, there were no observable differences in vascularity of platelet-poor plasma, monocyte, and granulocyte injections. We attribute this to the lack of VEGF in any of these preparations. The aforementioned study also showed that the lack of platelets in injection was enough to prevent the angiogenic effect of this treatment.³⁵

Continue to: The goal of this study was...

 

 

The goal of this study was to assess the morphology, cellularity, and vascularity of normal tendons after injections of different leukocyte populations. This is clinically important because of the potential to tailor future PRP injections on a patient-by-patient basis. In patients requiring an anabolic response, leukocyte-poor PRP may be the best option. In contrast, when patient pathology requires an inflammatory response to improve healing³⁶ or breakdown fibrotic tissue, as seen in tendinosis, leukocyte-rich PRP may be warranted. Further, properly controlled clinical studies are needed to validate these recommendations.

Limitations inherent in this study are those similar to other in vivo studies. First, the results of injections into rat tendons may not be translatable to human tendons. Despite this limitation, the rat is a common model for tendon research.³¹ A second limitation is that this study injected healthy Achilles tendons, rather than tendons with preexisting tendinopathy. In a naturally occurring tendinopathy, there may be other factors present that interact with PRP, and this model negates the contribution of these factors. Finally, while the IHC and morphological data show clear changes, the cellularity data are not clear in identifying the type of cells that were increased by granulocyte injection. However, the cells appeared rounded, resembling inflammatory infiltrate; a common cell type seen in tendons.² While fibroblasts are also a common infiltrate during chronic tendinopathy, they are generally flat and appear on H&E as long spindle shaped cells. The last limitation of this study is the lack of functional mechanical testing since, clinically, healing of the tendon is also related to the strength of the tendon. Thus, we believe the increased cellularity of the tendons after granulocyte injections is representative of an increase in inflammation. Moreover, our results are consistent with the increased inflammation previously reported linking leukocytes to tendon inflammation.^20,22,32 It is interesting to note that the increase in inflammation does not lead to an increase in vascularity as could be expected.

CONCLUSION

We found that the injection of leukocytes into healthy rat Achilles tendons increases inflammation, as evidenced by increased cellularity and disrupted morphology, which suggests that leukocyte-rich PRP preparations may be contraindicated in settings of acute tendonitis. However, these preparations may be useful for a specific subset of tendinopathies, including chronic tendinosis.

ABSTRACT

In this study, we determine the in vivo effects of injecting sub-populations of leukocytes into normal rat Achilles tendons via a controlled laboratory study. Allogenic monocytes, granulocytes, or plasma were injected into 24 healthy rat Achilles tendons. Treated and contralateral un-treated control tendons then assessed for cellularity, histologic morphology, and vascularity after 7 and 14 days. Significant increases of 221% and 249% in cellularity (P = 0.014) were seen on day 14 within Achilles tendons injected with granulocytes as compared to plasma and monocytes, respectively. Also, significant improvement in morphology (P = 0.029) between days 7 and 14 was seen for the granulocyte injected Achilles tendons. Significant increases in cellularity after an injection of granulocytes, compared to monocytes and plasma, corresponds to a significant increase in inflammation within the tissue, suggesting that leukocyte-rich platelet-rich plasma (PRP) preparations are proinflammatory and potentially catabolic when injected into tendon tissue. The concentration and composition of white blood cells within PRP preparations is variable and needs to be better understood in order to optimize clinical utility of PRP injections.

Continue to: Tendinopathies are debilitating conditions...

Tendinopathies are debilitating conditions affecting patients worldwide every day. They arise most frequently from tendon overuse resulting in pathology.¹ There are 2 major subtypes of tendinopathy: tendinosis and tendinitis. Tendinosis, the more common condition, is characterized by long-term, chronic degradation of tendon tissue resulting in fibrosis from infiltrating fibroblasts.² Tendinitis, the less common condition, is characterized by an acute inflammatory response and inflammatory cell infiltrate.² Both conditions are common, with Achilles tendinopathy affecting 11% of runners and lateral epicondylitis affecting 1% to 3% of the general population.^3,4 Many sports-related overuse injuries, such as tendinopathies, go undiagnosed for extended periods of time because medical attention is avoided in order to prevent time loss from training or competing.⁵ These delays could be eliminated if a non-surgical option for treating tendon pathology was available.

Tendinopathies are believed to result from tendon overuse that causes micro-damage to collagen, as well as from significant changes in protein and enzyme composition within the tendon.⁶ The damage accumulates over time and eventually leads to chronic inflammation or fibrotic change within tendons, in both cases weakening the tendon and causing pain. Currently, accepted treatments for tendinopathies include: nonsteroidal anti-inflammatory drugs, physical therapy, ultrasound, laser-therapy, corticosteroids, glyceryl trinitrate patches, extracorporeal shock wave therapy, sclerotherapy, and surgery.⁷ Recently, platelet-rich plasma (PRP) therapy has emerged as a promising treatment for tendinopathies, as well as a variety of other orthopedic indications.

PRP consists of autologous blood from the patient, centrifuged to increase the amount of platelets in the sample above baseline, and subsequently injected around an affected tendon or joint.⁸ PRP is used to treat tendinopathy because it can supply injured tendons with blood components that aid in healing, which tendons do not receive due to poor vascularity.⁹ These components include growth factors, such as platelet derived growth factor (PDGF), transforming growth factor-β (TGF-β), vascular endothelial growth factor (VEGF), endothelial growth factor, and leukocytes that can stimulate an inflammatory response within the injured tissue.¹⁰ The inflammatory response from the PRP induces a more robust reconstruction and revascularization of the injured tissue, stimulating proliferation, and remodeling.^11,12However, significant variability exists within the platelets, leukocytes, and growth factors that comprise PRP. This is attributed to 3 major causes. First, current commercial preparations of PRP result in differing platelet concentrations, as well as leukocyte-rich and leukocyte-poor compositions.^13,14 Variability in platelet concentrations results in unreliable amounts of growth factors, including cytokines, TGF-β, PDGF, VEGF and basic fibroblast growth factor in each preparation, while leukocyte levels affect inflammation, all leading to variable effects for each preparation.^15,16Second, despite sex and age of the PRP donor not being significant factors influencing variation in growth factor concentrations, the existence of an unexplained variation in concentrations of growth factors between different donors has been observed.¹⁷ Third, the selection of activating agents, bovine thrombin or calcium chloride, and their application, whether to the elbow, shoulder, or knee, produces variability.¹⁸

While the effects of platelets and growth factors in PRP have been well studied, less is known about the effects of differing cell types. Recently it was reported that the concentrations of leukocytes directly affect the outcomes of PRP injections. McCarrel and colleagues^19,20 found that as the number of leukocytes increased, there was a concomitant increase in the expression of inflammatory cytokines and catabolic activity. This effect may result in inferior healing of injured tissues and is attributed to the release of pro-inflammatory cytokines such as interleukin-1β from the leukocytes.²¹ There is also evidence that minimizing the catabolic effect of leukocytes may be just as important to tissue healing as the maximizing anabolic effect of platelets and growth factors.²²

The use of PRP has been highly disputed in recent years due to conflicting reports of its success in treating orthopedic conditions. Numerous favorable studies have shown benefit for treating chronic and acute orthopedic injuries including; rotator cuff tear repair, chronic refractory patellar tendinopathy, and chronic lateral tendinosis/epicondylitis.^23-26 Concurrently, articles demonstrating no significant effects from PRP have also been published. One study claiming that PRP injections did not improve outcomes of chronic Achilles tendinopathy did not differentiate whether patients had tendinosis or tendinitis, and did not consider leukocyte concentration in their PRP preparations²⁷ Another study that determined PRP is not beneficial to the healing of ruptured Achilles tendons after surgical repair also failed to consider the concentration of leukocytes in their PRP preparations.²⁸ One of the difficulties in comparing these studies is their heterogeneous nature. This arises from the use of different conditions in each study that makes the studies incomparable. Variations in PRP preparations lead to different concentrations of growth factors, platelets, and leukocyte concentrations. Additionally, tendinopathy models were not specified as tendinosis and tendonitis, and models or patients were not controlled for age, sex, or comorbidities. Given that leukocyte-rich and leukocyte-poor PRP preparations are currently widely used in clinical practice, the discovery of which type of preparation is indicated in which setting is paramount to evidence-based use of this treatment modality. Due to reports suggesting that leukocytes may be detrimental to tendon healing, determining which types of leukocytes are responsible for these effects is vital. As such, the purpose of this study is to determine the in vivo effects of sub-populations of leukocytes on normal rat tendons. This study design allowed us to isolate the effects of the injections to induce a response and remove confounding effects of normal healing response to a damaged tendon and effects from the injection itself. Our hypothesis was that the injection of leukocytes would cause an inflammatory response in rat tendons, leading to catabolic outcomes.

Continue to: METHODS...

 

 

METHODS

This was a prospective, in vivo, placebo controlled, randomized animal study. The University’s Institutional Animal Care and Use Committee approved all procedures prior to initiation. Twenty-four male Sprague-Dawley rats were randomized to 3 treatment groups (n = 8): monocytes; granulocytes, and; plasma, as a negative control.

Allogenic blood from 6 additional rats was collected into K2EDTA tubes via cardiac puncture. Allogenic, as opposed to autogenic, blood is commonly used in rat models because of low immunogenic response to blood from rats of the same strain and litter.^29,30 The blood was then pooled and the red cells lysed by incubation with Red Blood Cell Lysis Buffer (Roche). The samples were then sorted into fractions containing monocytes and granulocytes using fluorescence activated cell sorting (FACS) using a FACSAria (BD Biosciences). Cells were sorted using Purified PE Mouse Anti-Rat CD11b/c antibodies (BD Pharmingen) specific to monocytes, granulocytes, macrophages, dendritic cells, and microglia, APC-Cy7 Mouse Anti-Rat CD45 antibodies (BD Pharmingen) specific to all hematopoietic cells except erythrocytes, and FITC Mouse Anti-Rat CD42d antibodies (BD Pharmingen) specific to megakaryocytes and platelets. 20 μL of 0.2 mg/mL CD11b/c, 20 μL of 0.2 mg/mL CD 45, and 10 μL of 0.5 mg/mL CD42d antibodies were added to 1 mL of condensed non-red cells collected from the 6 rats and incubated at room temperature in the dark for 15 minutes. A fraction containing only platelet-poor plasma was also collected. For all treatments the injection volume was 75 μL. Rats in the monocyte group were injected with 200,000 cells in platelet-poor plasma, those in the granulocyte group were injected with 900,000 cells in platelet-poor plasma, and rats in the plasma control group received only platelet-poor plasma. The cell concentrations were based on previous studies that documented these concentrations that are found in typical leukocyte-rich PRP preparations.¹³

The animals were anesthetized with isoflurane gas and then injected aseptically once into their right Achilles tendon. The left Achilles tendon was used as an un-injected control, giving a total of 48 total Achilles tendons studied. At days 7 and 14 post-injection, 4 rats from each group were euthanized and the Achilles tendons were harvested.

The tendons were fixed in neutral buffered formalin for 24 hours and then embedded in paraffin and sectioned sagittally at 12 μm. The tendons were then stained with hematoxylin and eosin (H&E) using standard histological protocols and examined by 3 individuals trained to assess cellularity and morphology. All samples were assigned unrecognizable numbers and randomized prior to examination by individuals. Cell counts were based on the number of nuclei present in 3 mid-tendon high-power fields (400x) per sample. Morphology was graded on a scale of 1 to 3, with 1 being a normal tendon and 3 having severe pathology with total loss of alignment and crimping on 3 low-power fields (100x) per sample (Figures 1A-1G).

Vascularity was assessed by immunohistochemical staining using Rabbit Polyclonal Anti-CD31 antibodies (Abcam), a marker for vascular endothelial cells, using a Vectastain ABC Kit (Vector Laboratories) system and the ImmPACT AEC Peroxidase (HRP) Substrate (Vector Laboratories). Following staining, automated image analysis was performed (Bioquant). Briefly, all areas that did not contain tendon were masked. CD31 positive areas were then quantified using global thresholding. Vascularity was then calculated as ratio of CD31 positive area to total tendon area. Analyses were performed on 3 mid-tendon medium-power (200x) fields per sample.

For cellularity and morphology, the results for the injected tendons were normalized to those of their contralateral untreated controls and reported as a percentage. Results for vascularity were compared directly between treated tendons. Differences were assessed between groups at each time-point using Independent Samples Median Tests. When significant differences were identified, pairwise comparisons were performed to identify the source of the differences. All analyses were conducted using SPSS (V22, SAS Institute) with significant differences determined for values of P < 0.05.

RESULTS

No significant differences in cellularity between groups were seen at day 7 (P = 0.368) (Figures 1A-1G). However, a significant difference in cellularity between groups was seen at day 14 (P = 0.014). Pairwise tests showed there to be a significant increase in the number of cells in the tendons treated with granulocytes from 221% and 249% in cellularity (P = 0.014) on day 14, as compared to both monocytes and plasma, respectively. Morphologically, no significant differences were seen between groups at either time-point (P = 0.091 for day 7 and P = 1.000 for day 14) (Figures 2A-2G). However, a significant improvement in morphology was observed from day 7 to day 14 in the granulocyte group from 60% to 165% (P = 0.029). Finally, no differences were seen in vascularity between treatment groups at either time-point (P = 0.368 for day 7 and P = 0.535 for day 14) (Figures 3A-3G).

Continue to: DISCUSSION...

 

 

DISCUSSION

Our hypothesis that the injection of leukocytes would cause an inflammatory response in rat tendons leading to catabolic outcomes was confirmed in the granulocyte group. It should be noted that prior to the catabolic outcome, there was a transient anabolic effect in the granulocyte group during the second week. Deterioration in morphology was observed in the tendons injected with granulocytes on day 7, which subsequently recovered in the following week. We found that injecting granulocytes into normal tendons resulted in an increase in inflammatory cellularity, when compared to monocytes and plasma injections. 

Limitations inherent in this study are those similar to other in vivo studies. To begin with, the results of injections into rat tendons may not be translatable to human tendons. Despite this limitation, the rat is a common model for tendon research.³¹ Another limitation is that this study injected healthy Achilles tendons, rather than tendons with preexisting tendinopathy. In a naturally occurring tendinopathy, there may be other factors present that interact with PRP, and this model negates the contribution of these factors. Finally, while the immunohistochemistry (IHC) and morphological data are clear, the cellularity data are not clear in identifying the type of cells that were increased by granulocyte injection.  However, the cells appeared rounded, resembling inflammatory infiltrate; a common cell type seen in tendons.² While fibroblasts are also a common infiltrate during chronic tendinopathy, they are generally flat and appear on H&E as long spindle shaped cells. Thus, we believe the increased cellularity of the tendons after granulocyte injections is representative of an increase in inflammation. The increased cellularity could be due to the increased number of cells injected into the tendon; however, our conclusions are consistent with the increased inflammation previously reported linking leukocytes to tendon inflammation.^20,22,32

In terms of morphology, we hypothesized that degenerative changes would be seen in the tendons that were injected with granulocytes due to the inflammatory action of these cells. As part of the granulocyte response, neutrophils release proteases and macrophages can stimulate collagen synthesis via fibroblasts, both causing change within the extracellular matrix.^33,34 Indeed, we observed a significant change in tissue morphology in the granulocyte group over the course of 14 days. As the degenerative and regenerative effects of granulocytes take time to present, this is likely what we observed to occur between day 7 and 14 after treatment. These observations are also consistent with prior observations that leukocyte-rich PRP injections can be detrimental to tendon healing, but beneficial to tissue degeneration in the setting of chronic tendonitis.²⁰

We hypothesized that the vascularity of the tendons would be similar in all preparations. This was based on previous studies demonstrating that the lack of platelets in the platelet-poor plasma fraction is sufficient to deplete VEGF, the angiogenic agent in PRP.³⁵ In this study, there were no observable differences in vascularity of platelet-poor plasma, monocyte, and granulocyte injections. We attribute this to the lack of VEGF in any of these preparations. The aforementioned study also showed that the lack of platelets in injection was enough to prevent the angiogenic effect of this treatment.³⁵

Continue to: The goal of this study was...

 

 

The goal of this study was to assess the morphology, cellularity, and vascularity of normal tendons after injections of different leukocyte populations. This is clinically important because of the potential to tailor future PRP injections on a patient-by-patient basis. In patients requiring an anabolic response, leukocyte-poor PRP may be the best option. In contrast, when patient pathology requires an inflammatory response to improve healing³⁶ or breakdown fibrotic tissue, as seen in tendinosis, leukocyte-rich PRP may be warranted. Further, properly controlled clinical studies are needed to validate these recommendations.

Limitations inherent in this study are those similar to other in vivo studies. First, the results of injections into rat tendons may not be translatable to human tendons. Despite this limitation, the rat is a common model for tendon research.³¹ A second limitation is that this study injected healthy Achilles tendons, rather than tendons with preexisting tendinopathy. In a naturally occurring tendinopathy, there may be other factors present that interact with PRP, and this model negates the contribution of these factors. Finally, while the IHC and morphological data show clear changes, the cellularity data are not clear in identifying the type of cells that were increased by granulocyte injection. However, the cells appeared rounded, resembling inflammatory infiltrate; a common cell type seen in tendons.² While fibroblasts are also a common infiltrate during chronic tendinopathy, they are generally flat and appear on H&E as long spindle shaped cells. The last limitation of this study is the lack of functional mechanical testing since, clinically, healing of the tendon is also related to the strength of the tendon. Thus, we believe the increased cellularity of the tendons after granulocyte injections is representative of an increase in inflammation. Moreover, our results are consistent with the increased inflammation previously reported linking leukocytes to tendon inflammation.^20,22,32 It is interesting to note that the increase in inflammation does not lead to an increase in vascularity as could be expected.

CONCLUSION

We found that the injection of leukocytes into healthy rat Achilles tendons increases inflammation, as evidenced by increased cellularity and disrupted morphology, which suggests that leukocyte-rich PRP preparations may be contraindicated in settings of acute tendonitis. However, these preparations may be useful for a specific subset of tendinopathies, including chronic tendinosis.

ABSTRACT

Quadriceps tendon ruptures disrupt the extensor mechanism of the knee and require urgent surgical management. Traditional repair techniques have had mixed biomechanical and clinical results risking weakness and extensor lag. We describe a novel technique using tape suture and knotless anchors, which has performed superiorly during biomechanical testing and yielded terrific early clinical results. 

Continue to: Quadriceps tendon rupture...

Quadriceps tendon rupture is an uncommon yet potentially devastating knee injury with an estimated incidence of 1.37 in 100,000.¹ It most often occurs in male, middle-aged or older patients with degenerative tendon changes and serious systemic diseases, such as chronic renal failure, diabetes mellitus, rheumatoid arthritis, and disorders requiring long-term steroid use (tissue quality is often compromised by patient age and comorbidities).^2-10 Whereas partial tears with an intact extensor mechanism may be managed nonoperatively, prompt operative intervention is indicated in cases of complete tear or an incompetent extensor mechanism to facilitate early range of motion (ROM) and return of knee function.^2-4,8,9

The standard of care is repair with a nonabsorbable suture passed through transosseous patellar tunnels, often with several weeks of postoperative immobilization to protect the repair.^3,4,7,10-12 Reported complications of this method include significant extension lag, decreased strength, and ROM compared with the contralateral knee, chronic pain, and iatrogenic patellar fracture.^8,13-18 Repair techniques using suture anchors have been proposed as viable alternatives, but biomechanical studies comparing them with standard transosseous repair have reported mixed results.^{7,10-12,18-20} Two studies found improved biomechanical characteristics with suture anchors,^10,21 but 2 others found the characteristics of suture anchor fixation equal to¹¹ or worse than¹² those of transosseous fixation. In light of the controversy regarding strength and clinical outcomes of suture anchor repair compared with transosseous repair, new and potentially superior surgical interventions should be considered.

We recently completed a cadaveric study comparing the biomechanical properties of a novel quadriceps tendon repair technique using 4.75-mm biocomposite knotless suture anchors with suture tape and the properties of conventional techniques using either transosseous or suture anchor repair alone.²² In the cadaveric model, compared with transosseous and fully threaded suture anchor techniques, repair of quadriceps tendon ruptures with this knotless suture anchor with suture tape technique was biomechanically superior in cyclic displacement, construct stiffness, and ultimate load to failure.²² Additionally, this method allows for less extensive dissection, shorter operative times, and the potential for earlier and more aggressive rehabilitation protocols.²² We propose this technique, presented in this article, as a superior alternative to traditional quadriceps tendon repair techniques.

TECHNIQUE

The patient is placed in supine position with a tourniquet placed on the proximal thigh. A midline incision is made from the proximal pole of the patella, proximally by 5 cm. A combination of sharp and blunt dissection is performed through skin and subcutaneous tissues down to the extensor mechanism, exposing the proximal pole of the patella and the torn quadriceps tendon.

The distal aspect of the quadriceps tendon is then débrided of any devitalized tissue and secured with an Allis clamp. A long tape suture (FiberTape; Arthrex) is then used to place a locking Krackow stitch in a distal-to-proximal and then proximal-to-distal direction for 5 throws in each direction within the quadriceps tendon, with the tails exiting distally at the tear site. Care is taken with each pass to ensure that there is no slack within the system.

Continue to: The proximal pole of the patella...

The proximal pole of the patella is then prepared by débriding any remaining soft tissue back to an area of exposed subcortical bone, which is débrided to a bleeding bony bed. Holes are drilled in the medial and lateral thirds of the patella at the proximal pole using the drill for 4.75-mm biocomposite knotless suture anchors (SwiveLock; Arthrex). The tap for the 4.75-mm anchors is then passed at each guide hole. In hard bone, double-tapping is recommended.

Next, the medial strand of tape suture is loaded within a 4.75-mm biocomposite knotless suture anchor eyelet and reduced to the patella. The medial anchor is malleted and screwed into place, while tension is kept on the lateral strand with the knee in full extension. The lateral strand is then placed into its 4.75-mm biocomposite knotless suture anchor, reduced to the patella, and then malleted and screwed into place in the lateral hole, thereby completing the core portion of the repair (Figures A-D). The core strands from the 4.75-mm biocomposite knotless suture anchors are then back-passed in mattress fashion and tied, and medial and lateral retinacular repairs are then performed using supersuture tape (SutureTape or FiberWire; Arthrex).

After surgery, the patient is placed in a knee brace locked in full extension and allowed to weight-bear as tolerated using crutches. During the first week, knee ROM is allowed up to 30°. During weeks 2 to 6 passive ROM is gradually increased to 90°, and use of crutches is tapered. At week 6 the brace is unlocked for ambulation; it may be discontinued after 7 to 8 weeks or when determined safe. Light activity is permitted from month 4 to month 6. A patient who achieves satisfactory strength, is clinically examined, and progresses through rehabilitation is allowed to return to fully unrestricted sport.

DISCUSSION

Quadriceps tendon rupture is an uncommon clinical entity that requires early surgical management.^1-5,12,17,19 The standard of care is passage of nonabsorbable sutures through transosseous patellar bone tunnels, but repair with suture anchors has been studied as an alternative that allows for less tissue trauma, decreased operative time, safe early initiation of rehabilitation protocols, and reduced risk of patella fracture or damage.^{3,7,10-12,18-20,21,23} Despite these potential advantages, biomechanical studies have yielded inconsistent results regarding the superiority of suture anchor repair over repair with transosseous tunnels.^{7,10-12,18-20} We propose quadriceps tendon repair using the 4.75-mm biocomposite knotless suture anchor with tape suture technique as a biomechanically superior alternative to either transosseous tunnels or suture anchor repair alone, with significant advantages both in and out of the operating room.

Results of biomechanical studies comparing transosseous tunnel repair and standard suture anchor repair have been mixed, though the heterogeneity of their study methods and endpoints makes direct comparisons difficult.^{7,10-12,18-20} Petri and colleagues¹⁰ and Sherman and colleagues²¹ reported statistically significant higher load to failure¹⁰ and reduced gapping during cyclic loading^10,21 with suture anchor repair relative to transosseous repair. However, Hart and colleagues¹² found that repair with suture anchors had lower ultimate tensile load, and they concluded that transosseous repair is superior. Lighthart and colleagues¹¹ found no significant difference in displacement between the 2 repairs.

Continue to: In our cadaveric biomechanical study...

In our cadaveric biomechanical study, a novel 4.75-mm biocomposite knotless suture anchor with suture tape repair was compared with traditional 3-tunnel transosseous repair and with standard 2-anchor suture anchor repair.²² Statistically significant superiority was found across multiple parameters, including initial tendon displacement, stiffness, and ultimate load to failure (vs 5.5-mm biocomposite fully threaded suture anchor repair), as well as initial and late tendon displacement, stiffness, and ultimate load to failure (vs transosseous repair).²² Although definitive conclusions are difficult to draw on the basis of prior cadaveric studies comparing standard suture anchor repair and transosseous repair, our results decidedly favor the biomechanical characteristics of this 4.75-mm biocomposite knotless suture anchor with suture tape repair and make it a potentially superior repair technique based on biomechanics alone.²²

Similarly to standard repair with suture anchors, repair using a 4.75-mm biocomposite knotless suture anchor with tape suture eliminates the need to expose the distal pole of the patella.^7,10-12,21 This allows for a smaller surgical incision, less extensive dissection, and prevents possible interference with the patellar tendon.^7,10-12,21 Additionally, it eliminates the risk of iatrogenic patellar fracture and damage to the articular surface from drilling the transpatellar tunnels.^17,18 Both our own review of cases repaired with our 4.75-mm biocomposite knotless suture anchor with suture tape technique as well as studies of suture anchor repair have consistently found operative times of <1 hour.²¹ Shorter operative times and smaller surgical wounds are advantageous given that many of these patients have medical comorbidities that predispose them to intraoperative and wound-healing complications.^12,19-22

Optimal rehabilitation protocols for quadriceps tendon repair are a matter of controversy. Multiple studies of repair with transosseous patellar tunnels describe immobilization for 6 weeks after surgery, but there has been a recent push toward early motion.^7,13,23,24 Reported complications of extended immobilization include limited flexion, pain, weakness, decreased patellar mobility, and patella baja.¹⁴ Studies have suggested that, while excessive loading can cause gap formation and weaken the repair, some controlled motion is necessary to heal the tendon^23,25 and reduce the risks of stiffness and atrophy.¹⁴ The improved biomechanical characteristics of the 4.75-mm biocomposite knotless suture anchor with tape suture technique allow for safe early initiation of ROM exercises and accelerated rehabilitation protocols.

In our early experience with this technique, functional outcomes have been excellent. A formal 2-year outcome study of patients who have undergone quadriceps tendon repair with this 4.75-mm biocomposite knotless suture anchor with tape suture technique is under way.

ABSTRACT

Quadriceps tendon ruptures disrupt the extensor mechanism of the knee and require urgent surgical management. Traditional repair techniques have had mixed biomechanical and clinical results risking weakness and extensor lag. We describe a novel technique using tape suture and knotless anchors, which has performed superiorly during biomechanical testing and yielded terrific early clinical results. 

Continue to: Quadriceps tendon rupture...

Quadriceps tendon rupture is an uncommon yet potentially devastating knee injury with an estimated incidence of 1.37 in 100,000.¹ It most often occurs in male, middle-aged or older patients with degenerative tendon changes and serious systemic diseases, such as chronic renal failure, diabetes mellitus, rheumatoid arthritis, and disorders requiring long-term steroid use (tissue quality is often compromised by patient age and comorbidities).^2-10 Whereas partial tears with an intact extensor mechanism may be managed nonoperatively, prompt operative intervention is indicated in cases of complete tear or an incompetent extensor mechanism to facilitate early range of motion (ROM) and return of knee function.^2-4,8,9

The standard of care is repair with a nonabsorbable suture passed through transosseous patellar tunnels, often with several weeks of postoperative immobilization to protect the repair.^3,4,7,10-12 Reported complications of this method include significant extension lag, decreased strength, and ROM compared with the contralateral knee, chronic pain, and iatrogenic patellar fracture.^8,13-18 Repair techniques using suture anchors have been proposed as viable alternatives, but biomechanical studies comparing them with standard transosseous repair have reported mixed results.^{7,10-12,18-20} Two studies found improved biomechanical characteristics with suture anchors,^10,21 but 2 others found the characteristics of suture anchor fixation equal to¹¹ or worse than¹² those of transosseous fixation. In light of the controversy regarding strength and clinical outcomes of suture anchor repair compared with transosseous repair, new and potentially superior surgical interventions should be considered.

We recently completed a cadaveric study comparing the biomechanical properties of a novel quadriceps tendon repair technique using 4.75-mm biocomposite knotless suture anchors with suture tape and the properties of conventional techniques using either transosseous or suture anchor repair alone.²² In the cadaveric model, compared with transosseous and fully threaded suture anchor techniques, repair of quadriceps tendon ruptures with this knotless suture anchor with suture tape technique was biomechanically superior in cyclic displacement, construct stiffness, and ultimate load to failure.²² Additionally, this method allows for less extensive dissection, shorter operative times, and the potential for earlier and more aggressive rehabilitation protocols.²² We propose this technique, presented in this article, as a superior alternative to traditional quadriceps tendon repair techniques.

TECHNIQUE

The patient is placed in supine position with a tourniquet placed on the proximal thigh. A midline incision is made from the proximal pole of the patella, proximally by 5 cm. A combination of sharp and blunt dissection is performed through skin and subcutaneous tissues down to the extensor mechanism, exposing the proximal pole of the patella and the torn quadriceps tendon.

The distal aspect of the quadriceps tendon is then débrided of any devitalized tissue and secured with an Allis clamp. A long tape suture (FiberTape; Arthrex) is then used to place a locking Krackow stitch in a distal-to-proximal and then proximal-to-distal direction for 5 throws in each direction within the quadriceps tendon, with the tails exiting distally at the tear site. Care is taken with each pass to ensure that there is no slack within the system.

Continue to: The proximal pole of the patella...

The proximal pole of the patella is then prepared by débriding any remaining soft tissue back to an area of exposed subcortical bone, which is débrided to a bleeding bony bed. Holes are drilled in the medial and lateral thirds of the patella at the proximal pole using the drill for 4.75-mm biocomposite knotless suture anchors (SwiveLock; Arthrex). The tap for the 4.75-mm anchors is then passed at each guide hole. In hard bone, double-tapping is recommended.

Next, the medial strand of tape suture is loaded within a 4.75-mm biocomposite knotless suture anchor eyelet and reduced to the patella. The medial anchor is malleted and screwed into place, while tension is kept on the lateral strand with the knee in full extension. The lateral strand is then placed into its 4.75-mm biocomposite knotless suture anchor, reduced to the patella, and then malleted and screwed into place in the lateral hole, thereby completing the core portion of the repair (Figures A-D). The core strands from the 4.75-mm biocomposite knotless suture anchors are then back-passed in mattress fashion and tied, and medial and lateral retinacular repairs are then performed using supersuture tape (SutureTape or FiberWire; Arthrex).

After surgery, the patient is placed in a knee brace locked in full extension and allowed to weight-bear as tolerated using crutches. During the first week, knee ROM is allowed up to 30°. During weeks 2 to 6 passive ROM is gradually increased to 90°, and use of crutches is tapered. At week 6 the brace is unlocked for ambulation; it may be discontinued after 7 to 8 weeks or when determined safe. Light activity is permitted from month 4 to month 6. A patient who achieves satisfactory strength, is clinically examined, and progresses through rehabilitation is allowed to return to fully unrestricted sport.

DISCUSSION

Quadriceps tendon rupture is an uncommon clinical entity that requires early surgical management.^1-5,12,17,19 The standard of care is passage of nonabsorbable sutures through transosseous patellar bone tunnels, but repair with suture anchors has been studied as an alternative that allows for less tissue trauma, decreased operative time, safe early initiation of rehabilitation protocols, and reduced risk of patella fracture or damage.^{3,7,10-12,18-20,21,23} Despite these potential advantages, biomechanical studies have yielded inconsistent results regarding the superiority of suture anchor repair over repair with transosseous tunnels.^{7,10-12,18-20} We propose quadriceps tendon repair using the 4.75-mm biocomposite knotless suture anchor with tape suture technique as a biomechanically superior alternative to either transosseous tunnels or suture anchor repair alone, with significant advantages both in and out of the operating room.

Results of biomechanical studies comparing transosseous tunnel repair and standard suture anchor repair have been mixed, though the heterogeneity of their study methods and endpoints makes direct comparisons difficult.^{7,10-12,18-20} Petri and colleagues¹⁰ and Sherman and colleagues²¹ reported statistically significant higher load to failure¹⁰ and reduced gapping during cyclic loading^10,21 with suture anchor repair relative to transosseous repair. However, Hart and colleagues¹² found that repair with suture anchors had lower ultimate tensile load, and they concluded that transosseous repair is superior. Lighthart and colleagues¹¹ found no significant difference in displacement between the 2 repairs.

Continue to: In our cadaveric biomechanical study...

In our cadaveric biomechanical study, a novel 4.75-mm biocomposite knotless suture anchor with suture tape repair was compared with traditional 3-tunnel transosseous repair and with standard 2-anchor suture anchor repair.²² Statistically significant superiority was found across multiple parameters, including initial tendon displacement, stiffness, and ultimate load to failure (vs 5.5-mm biocomposite fully threaded suture anchor repair), as well as initial and late tendon displacement, stiffness, and ultimate load to failure (vs transosseous repair).²² Although definitive conclusions are difficult to draw on the basis of prior cadaveric studies comparing standard suture anchor repair and transosseous repair, our results decidedly favor the biomechanical characteristics of this 4.75-mm biocomposite knotless suture anchor with suture tape repair and make it a potentially superior repair technique based on biomechanics alone.²²

Similarly to standard repair with suture anchors, repair using a 4.75-mm biocomposite knotless suture anchor with tape suture eliminates the need to expose the distal pole of the patella.^7,10-12,21 This allows for a smaller surgical incision, less extensive dissection, and prevents possible interference with the patellar tendon.^7,10-12,21 Additionally, it eliminates the risk of iatrogenic patellar fracture and damage to the articular surface from drilling the transpatellar tunnels.^17,18 Both our own review of cases repaired with our 4.75-mm biocomposite knotless suture anchor with suture tape technique as well as studies of suture anchor repair have consistently found operative times of <1 hour.²¹ Shorter operative times and smaller surgical wounds are advantageous given that many of these patients have medical comorbidities that predispose them to intraoperative and wound-healing complications.^12,19-22

Optimal rehabilitation protocols for quadriceps tendon repair are a matter of controversy. Multiple studies of repair with transosseous patellar tunnels describe immobilization for 6 weeks after surgery, but there has been a recent push toward early motion.^7,13,23,24 Reported complications of extended immobilization include limited flexion, pain, weakness, decreased patellar mobility, and patella baja.¹⁴ Studies have suggested that, while excessive loading can cause gap formation and weaken the repair, some controlled motion is necessary to heal the tendon^23,25 and reduce the risks of stiffness and atrophy.¹⁴ The improved biomechanical characteristics of the 4.75-mm biocomposite knotless suture anchor with tape suture technique allow for safe early initiation of ROM exercises and accelerated rehabilitation protocols.

In our early experience with this technique, functional outcomes have been excellent. A formal 2-year outcome study of patients who have undergone quadriceps tendon repair with this 4.75-mm biocomposite knotless suture anchor with tape suture technique is under way.

ABSTRACT

Quadriceps tendon ruptures disrupt the extensor mechanism of the knee and require urgent surgical management. Traditional repair techniques have had mixed biomechanical and clinical results risking weakness and extensor lag. We describe a novel technique using tape suture and knotless anchors, which has performed superiorly during biomechanical testing and yielded terrific early clinical results. 

Continue to: Quadriceps tendon rupture...

Quadriceps tendon rupture is an uncommon yet potentially devastating knee injury with an estimated incidence of 1.37 in 100,000.¹ It most often occurs in male, middle-aged or older patients with degenerative tendon changes and serious systemic diseases, such as chronic renal failure, diabetes mellitus, rheumatoid arthritis, and disorders requiring long-term steroid use (tissue quality is often compromised by patient age and comorbidities).^2-10 Whereas partial tears with an intact extensor mechanism may be managed nonoperatively, prompt operative intervention is indicated in cases of complete tear or an incompetent extensor mechanism to facilitate early range of motion (ROM) and return of knee function.^2-4,8,9

The standard of care is repair with a nonabsorbable suture passed through transosseous patellar tunnels, often with several weeks of postoperative immobilization to protect the repair.^3,4,7,10-12 Reported complications of this method include significant extension lag, decreased strength, and ROM compared with the contralateral knee, chronic pain, and iatrogenic patellar fracture.^8,13-18 Repair techniques using suture anchors have been proposed as viable alternatives, but biomechanical studies comparing them with standard transosseous repair have reported mixed results.^{7,10-12,18-20} Two studies found improved biomechanical characteristics with suture anchors,^10,21 but 2 others found the characteristics of suture anchor fixation equal to¹¹ or worse than¹² those of transosseous fixation. In light of the controversy regarding strength and clinical outcomes of suture anchor repair compared with transosseous repair, new and potentially superior surgical interventions should be considered.

We recently completed a cadaveric study comparing the biomechanical properties of a novel quadriceps tendon repair technique using 4.75-mm biocomposite knotless suture anchors with suture tape and the properties of conventional techniques using either transosseous or suture anchor repair alone.²² In the cadaveric model, compared with transosseous and fully threaded suture anchor techniques, repair of quadriceps tendon ruptures with this knotless suture anchor with suture tape technique was biomechanically superior in cyclic displacement, construct stiffness, and ultimate load to failure.²² Additionally, this method allows for less extensive dissection, shorter operative times, and the potential for earlier and more aggressive rehabilitation protocols.²² We propose this technique, presented in this article, as a superior alternative to traditional quadriceps tendon repair techniques.

TECHNIQUE

The patient is placed in supine position with a tourniquet placed on the proximal thigh. A midline incision is made from the proximal pole of the patella, proximally by 5 cm. A combination of sharp and blunt dissection is performed through skin and subcutaneous tissues down to the extensor mechanism, exposing the proximal pole of the patella and the torn quadriceps tendon.

The distal aspect of the quadriceps tendon is then débrided of any devitalized tissue and secured with an Allis clamp. A long tape suture (FiberTape; Arthrex) is then used to place a locking Krackow stitch in a distal-to-proximal and then proximal-to-distal direction for 5 throws in each direction within the quadriceps tendon, with the tails exiting distally at the tear site. Care is taken with each pass to ensure that there is no slack within the system.

Continue to: The proximal pole of the patella...

The proximal pole of the patella is then prepared by débriding any remaining soft tissue back to an area of exposed subcortical bone, which is débrided to a bleeding bony bed. Holes are drilled in the medial and lateral thirds of the patella at the proximal pole using the drill for 4.75-mm biocomposite knotless suture anchors (SwiveLock; Arthrex). The tap for the 4.75-mm anchors is then passed at each guide hole. In hard bone, double-tapping is recommended.

Next, the medial strand of tape suture is loaded within a 4.75-mm biocomposite knotless suture anchor eyelet and reduced to the patella. The medial anchor is malleted and screwed into place, while tension is kept on the lateral strand with the knee in full extension. The lateral strand is then placed into its 4.75-mm biocomposite knotless suture anchor, reduced to the patella, and then malleted and screwed into place in the lateral hole, thereby completing the core portion of the repair (Figures A-D). The core strands from the 4.75-mm biocomposite knotless suture anchors are then back-passed in mattress fashion and tied, and medial and lateral retinacular repairs are then performed using supersuture tape (SutureTape or FiberWire; Arthrex).

After surgery, the patient is placed in a knee brace locked in full extension and allowed to weight-bear as tolerated using crutches. During the first week, knee ROM is allowed up to 30°. During weeks 2 to 6 passive ROM is gradually increased to 90°, and use of crutches is tapered. At week 6 the brace is unlocked for ambulation; it may be discontinued after 7 to 8 weeks or when determined safe. Light activity is permitted from month 4 to month 6. A patient who achieves satisfactory strength, is clinically examined, and progresses through rehabilitation is allowed to return to fully unrestricted sport.

DISCUSSION

Quadriceps tendon rupture is an uncommon clinical entity that requires early surgical management.^1-5,12,17,19 The standard of care is passage of nonabsorbable sutures through transosseous patellar bone tunnels, but repair with suture anchors has been studied as an alternative that allows for less tissue trauma, decreased operative time, safe early initiation of rehabilitation protocols, and reduced risk of patella fracture or damage.^{3,7,10-12,18-20,21,23} Despite these potential advantages, biomechanical studies have yielded inconsistent results regarding the superiority of suture anchor repair over repair with transosseous tunnels.^{7,10-12,18-20} We propose quadriceps tendon repair using the 4.75-mm biocomposite knotless suture anchor with tape suture technique as a biomechanically superior alternative to either transosseous tunnels or suture anchor repair alone, with significant advantages both in and out of the operating room.

Results of biomechanical studies comparing transosseous tunnel repair and standard suture anchor repair have been mixed, though the heterogeneity of their study methods and endpoints makes direct comparisons difficult.^{7,10-12,18-20} Petri and colleagues¹⁰ and Sherman and colleagues²¹ reported statistically significant higher load to failure¹⁰ and reduced gapping during cyclic loading^10,21 with suture anchor repair relative to transosseous repair. However, Hart and colleagues¹² found that repair with suture anchors had lower ultimate tensile load, and they concluded that transosseous repair is superior. Lighthart and colleagues¹¹ found no significant difference in displacement between the 2 repairs.

Continue to: In our cadaveric biomechanical study...

In our cadaveric biomechanical study, a novel 4.75-mm biocomposite knotless suture anchor with suture tape repair was compared with traditional 3-tunnel transosseous repair and with standard 2-anchor suture anchor repair.²² Statistically significant superiority was found across multiple parameters, including initial tendon displacement, stiffness, and ultimate load to failure (vs 5.5-mm biocomposite fully threaded suture anchor repair), as well as initial and late tendon displacement, stiffness, and ultimate load to failure (vs transosseous repair).²² Although definitive conclusions are difficult to draw on the basis of prior cadaveric studies comparing standard suture anchor repair and transosseous repair, our results decidedly favor the biomechanical characteristics of this 4.75-mm biocomposite knotless suture anchor with suture tape repair and make it a potentially superior repair technique based on biomechanics alone.²²

Similarly to standard repair with suture anchors, repair using a 4.75-mm biocomposite knotless suture anchor with tape suture eliminates the need to expose the distal pole of the patella.^7,10-12,21 This allows for a smaller surgical incision, less extensive dissection, and prevents possible interference with the patellar tendon.^7,10-12,21 Additionally, it eliminates the risk of iatrogenic patellar fracture and damage to the articular surface from drilling the transpatellar tunnels.^17,18 Both our own review of cases repaired with our 4.75-mm biocomposite knotless suture anchor with suture tape technique as well as studies of suture anchor repair have consistently found operative times of <1 hour.²¹ Shorter operative times and smaller surgical wounds are advantageous given that many of these patients have medical comorbidities that predispose them to intraoperative and wound-healing complications.^12,19-22

Optimal rehabilitation protocols for quadriceps tendon repair are a matter of controversy. Multiple studies of repair with transosseous patellar tunnels describe immobilization for 6 weeks after surgery, but there has been a recent push toward early motion.^7,13,23,24 Reported complications of extended immobilization include limited flexion, pain, weakness, decreased patellar mobility, and patella baja.¹⁴ Studies have suggested that, while excessive loading can cause gap formation and weaken the repair, some controlled motion is necessary to heal the tendon^23,25 and reduce the risks of stiffness and atrophy.¹⁴ The improved biomechanical characteristics of the 4.75-mm biocomposite knotless suture anchor with tape suture technique allow for safe early initiation of ROM exercises and accelerated rehabilitation protocols.

In our early experience with this technique, functional outcomes have been excellent. A formal 2-year outcome study of patients who have undergone quadriceps tendon repair with this 4.75-mm biocomposite knotless suture anchor with tape suture technique is under way.