Early healing of flexor tendon insertion site injuries: Tunnel repair is mechanically and histologically inferior to surface repair in a canine model.

Orthopedic injuries often require surgical reattachment of tendon to bone. Tendon ends can be sutured to bone by direct apposition to the bone surface or by placement within a bone tunnel. Our objective was to compare early healing of a traditional surface versus a novel tunnel method for repair of the flexor digitorum profundus (FDP) tendon insertion site in a canine model. A total of 70 tendon-bone specimens were analyzed 0, 5, 10 or 21 days after injury and repair, using tensile and range of motion mechanical testing, histology and densitometry. Ultimate force (a measure of repair strength) did not differ between surface and tunnel repairs at day 0. Both repair types had reduced strength at 10 and 21 days compared to 0 days, indicative of deterioration of suture grasping strength (tendon softening). At 21 days, tendons repaired in a bone tunnel had 38% lower ultimate force compared to surface repairs (p = 0.017). Histological findings were comparable between repair groups at 5 and 10 days but differed at 21 days, when we saw evidence of maturation of the tendon-bone interface in the surface repairs compared to an immature fibrous interface with no evidence of tendon-bone integration in the tunnel repairs. After accounting for bone removed by the tunnel, no difference in bone mineral density or trabecular bone volume existed between surface and tunnel repairs. If the results of our animal study extend to healing of the human FDP insertion, they indicate that FDP tendons should be reattached to the distal phalanx by suture to the cortical surface rather than suture in a bone tunnel.

Does subchondral bone affect the fate of osteochondral allografts during storage?

BACKGROUND: Osteochondral allografts currently are hypothermically stored for a minimum of 14 days to a maximum of 28 days before surgical implantation, making storage conditions increasingly important. Previous studies have suggested that graft deterioration during storage may result from degradative factors and residual marrow elements in the subchondral bone. HYPOTHESIS: Allografts stored with large bone-to-cartilage ratios will be compromised after prolonged storage compared with grafts with minimal bone. STUDY DESIGN: Controlled laboratory study. METHODS: Osteochondral plugs were harvested from 16 fresh human femoral condyles and randomly assigned to 1 of 3 groups based on bone-to-cartilage ratios: 1:1, 5:1, or 10:1. These ratios were considered on the basis that the 1:1 ratio is the minimum bone necessary to press-fit an allograft and 10:1 is the present ratio used by tissue banks for allograft storage. After 14 and 28 days of storage at 4 degrees C, the specimens were assessed for viability and viable cell density using confocal microscopy, proteoglycan synthesis by (35)SO4 incorporation, and glycosaminoglycan content. RESULTS: All grafts underwent a significant decline in viable cell density, proteoglycan synthesis, and chondrocyte viability (particularly in the superficial region) after 14 days of storage, but no differences were observed between the 1:1, 5:1, or 10:1 ratio groups at either day 14 or day 28. In addition, no significant difference was noted in the glycosaminoglycan content in any of the groups. CONCLUSION: Osteochondral allografts stored with a 10:1 bone-to-cartilage ratio, similar to tissue-banking ratios, performed no worse than allografts stored with minimal bone, suggesting that the bone-to-cartilage ratio plays little to no role in the degradation of allografts during prolonged storage. CLINICAL RELEVANCE: As the practice of osteochondral allograft resurfacing becomes more commonplace, it is important that surgeons understand the factors that affect graft quality.

Prolonged storage of osteochondral allografts: does the addition of fetal bovine serum improve chondrocyte viability?

Osteochondral plugs were harvested from eight fresh human femoral condyles within 96 hours of donor death. The plugs were either stored in a serum-free media containing glucose, salts, and amino acids or 10% fetal bovine serum at 4 degrees C. After 28 days of storage, the osteochondral plugs were analyzed for chondrocyte viability and viable cell density using confocal microscopy, proteoglycan synthesis by (35)SO4 incorporation, and glycosaminoglycan content. Chondrocyte viability and cell density were significantly lower in grafts stored in serum-free media compared to fetal bovine serum, 27% versus 68% (P < .001) and 3250 cells/mm3 versus 8960 cells/mm3, respectively (P < .001). The metabolic activity determined by proteoglycan synthesis was significantly better in the specimens stored in fetal bovine serum (P < .01). No significant difference was detected between the glycosaminoglycan content in any of the specimens. These data suggest that the quality of osteochondral allografts as measured by chondrocyte viability, viable cell density, and proteoglycan synthesis is superior after storage in fetal bovine serum versus serum-free media. These results must be taken cautiously, however, as the clinical ramifications of storage in fetal bovine serum, including potential infectious disease transmission risks and immunogenic factors, have yet to be studied.

Analysis of stored osteochondral allografts at the time of surgical implantation.

BACKGROUND: To date, the morphological, biochemical, and biomechanical characteristics of articular cartilage in osteochondral allografts that have been stored have not been fully described. HYPOTHESIS: Osteochondral allografts procured and stored commercially for a standard period as determined by tissue banking protocol will have compromised chondrocyte viability but preserved extracellular matrix quality. STUDY DESIGN: Controlled laboratory study. METHODS: Unused cartilage from 16 consecutive osteochondral allografts was sampled during surgery after tissue bank processing and storage. Ten grafts were examined for cell viability and viable cell density using confocal microscopy, proteoglycan synthesis via 35SO4 uptake, and glycosaminoglycan content and compared with fresh cadaveric articular cartilage. Biomechanical assessment was performed on the 6 remaining grafts by measuring the indentation stiffness of the cartilage. RESULTS: The mean storage time for the transplanted specimens was 20.3 +/- 2.9 days. Chondrocyte viability, viable cell density, and 35SO4 uptake were significantly lower in allografts at implantation when compared to fresh, unstored controls, whereas matrix characteristics, specifically glycosaminoglycan content and biomechanical measures, were unchanged. In addition, chondrocyte viability in the stored allografts was preferentially decreased in the superficial zone of cartilage. CONCLUSION: Human osteochondral allografts stored for a standard period (approximately 3 weeks) before implantation undergo decreases in cell viability, especially in the critically important superficial zone, as well as in cell density and metabolic activity, whereas matrix and biomechanical characteristics appear conserved. The exact clinical significance of these findings, however, is unknown, as there are no prospective studies examining clinical outcomes using grafts stored for extended periods. CLINICAL RELEVANCE: Surgeons who perform this procedure should understand the cartilage characteristics of the graft after 21 days of commercial storage in serum-free media.

The insertion site of the canine flexor digitorum profundus tendon heals slowly following injury and suture repair.

Treatment of injuries of the flexor digitorum profundus (FDP) tendon insertion site has changed little during the past 50 years, in part because there are no reports describing flexor tendon insertion site healing. Our objective was to assess the effects of repair technique and post-operative time on tendon-bone healing using a canine model of injury and repair. We transected 48 FDP tendons from 24 dogs at their insertions and repaired them using either a four- or eight-strand suture technique. We assessed the mechanical properties of the repaired tendon-bone construct, tendon collagen biochemistry, and distal phalanx bone mineral density (BMD) at 0, 10, 21 and 42 days. Suture method had no significant effect on any outcome (p > 0.05). In particular, use of an eight-strand double modified Kessler technique did not result in increased stiffness or strength compared to a four-strand technique. With time, the repair site became stiffer, as demonstrated by a 230% increase in rigidity and a 50% decrease in strain from 0 to 42 days. However, from 0 to 42 days the ultimate force of the insertion site did not increase. This lack of increase in ultimate force was consistent with decreases in collagen content, non-reducible crosslinks and distal phalanx BMD. Taken together, our results indicate that the canine FDP tendon heals slowly after it is injured at its insertion site and sutured onto the distal phalanx. While these findings may be limited to the particular repair method we used, they demonstrate a need for devising new treatment strategies to improve healing of flexor tendon insertion site injuries.

Etanercept enhances preservation of osteochondral allograft viability.

BACKGROUND: Osteochondral allografts are an increasingly popular treatment for the repair of articular cartilage lesions. Current tissue bank protocols require bacteriological testing that takes from 21 to 28 days to process. During this time, tumor necrosis factor-alpha (TNF-α, a proapoptotic cytokine) is upregulated, resulting in loss of chondrocyte viability. To date, etanercept (a cytokine inhibitor) has not been studied in the current storage paradigm with the intention of preserving cell viability. PURPOSE: This study was undertaken to assess whether the addition of etanercept can improve the chondrocyte viability ofosteochondral allograft during storage. STUDY DESIGN: Controlled laboratory study. METHODS: Osteochondral allografts were harvested from 8 Boer goat femurs and placed into storage media and stored at 4°C for 28 days. The experimental group was supplemented with 10 µg/mL of etanercept. After storage, cell viability was assessed by live/dead staining and confocal microscopy. Specimens were also analyzed histologically and underwent histomorphologic analysis. TNF-α expression was measured with semiquantitative polymerase chain reaction. RESULTS: At 28 days, the percentage viability of the superficial zone in etanercept-treated allografts was maintained at significantly higher levels than those measured in the untreated group (69.3 ± 9.4 compared with 47.8 ± 19.1, P = .01). No difference was found histologically between the etanercept and the untreated group (ie, safranin O staining for glycosaminoglycan expression). Histomorphologic assessment showed no difference in indentation stiffness or roughness between groups. TNF-α expression was significantly decreased in the etanercept group compared to the untreated group. CONCLUSION: Etanercept was able to maintain cell viability of osteochondral allografts significantly better than the current storage paradigm after 28 days of storage. CLINICAL RELEVANCE: Maintaining the viability of the superficial zone will benefit outcomes by facilitating joint articulation via improved lubrication. Additionally, maintaining the cellular viability for increased periods of time may allow a greater window of time in which a suitable recipient may be found.

Variation of mesenchymal cells in polylactic acid scaffold in an osteochondral repair model.

OBJECTIVE: To achieve osteochondral regeneration utilizing transplantation of cartilage-lineage cells and adequate scaffolds, it is essential to characterize the behavior of transplanted cells in the repair process. The objectives of this study were to elucidate the survival of mesenchymal cells (MCs). In a polylactic acid (PLA) scaffold and assess the possibility of MC/PLA constructs for osteochondral repair. DESIGN: Bone marrow from mature male rabbits was cultured for 2 weeks, and fibroblast-like MCs, which contain mesenchymal stem cells (MSCs), were obtained. A cell/scaffold construct was prepared with one million MCs and a biodegradable PLA core using a rotator device. One week after culturing, the construct was transplanted into an osteochondral defect in the medial femoral condyle of female rabbits and the healing process examined histologically. To examine the survivability of transplanted MCs, the male-derived sex-determining region Y (SRY) gene was assessed as a marker of MCs in the defect by polymerase chain reaction (PCR). RESULTS: In the groups of defects without any treatment, and the transplantation of PLA without cells, the defects were not repaired with hyaline cartilage. The cartilaginous matrix by safranin O staining and type II collagen by immunohistochemical staining were recognized, however the PLA matrix was still present in the defects at 24 weeks after transplantation of the construct. During the time passage, transplanted MCs numbers decreased from 7.8 x 105 at 1 week, to 3.5 x 105 at 4 weeks, and to 3.8 x 104 at 12 weeks. Transplanted MCs were not detectable at 24 weeks. CONCLUSIONS: MCs contribute to the osteochondral repair expressing the cartilaginous matrix, however the number of MCs were decreasing with time (i.e. 24 weeks). These results could be essential for achieving cartilage regeneration by cell transplantation strategies with growth factors and/or gene therapy.

Enhanced flexor tendon healing through controlled delivery of PDGF-BB.

A fibrin/heparin-based delivery system was used to provide controlled delivery of platelet derived growth factor BB (PDGF-BB) in an animal model of intrasynovial flexor tendon repair. We hypothesized that PDGF-BB, administered in this manner, would stimulate cell proliferation and matrix remodeling, leading to improvements in the sutured tendon's functional and structural properties. Fifty-six flexor digitorum profundus tendons were injured and repaired in 28 dogs. Three groups were compared: (1) controlled delivery of PDGF-BB using a fibrin/heparin-based delivery system; (2) delivery system carrier control; and (3) repair- only control. The operated forelimbs were treated with controlled passive motion rehabilitation. The animals were euthanized at 7, 14, and 42 days, at which time the tendons were assessed using histologic (hyaluronic acid content, cellularity, and inflammation), biochemical (total DNA and reducible collagen crosslink levels), and biomechanical (gliding and tensile properties) assays. We found that cell activity (as determined by total DNA, collagen crosslink analyses, and hyaluronic acid content) was accelerated due to PDGF-BB at 14 days. Proximal interphalangeal joint rotation and tendon excursion (i.e., tendon gliding properties) were significantly higher for the PDGF-BB-treated tendons compared to the repair-alone tendons at 42 days. Improvements in tensile properties were not achieved, possibly due to suboptimal release kinetics or other factors. In conclusion, PDGF-BB treatment consistently improved the functional but not the structural properties of sutured intrasynovial tendons through 42 days following repair.

Role of apoptotic and matrix-degrading genes in articular cartilage and meniscus of mature and aged rabbits during development of osteoarthritis.

OBJECTIVE: To determine expression patterns of apoptotic and matrix-degrading genes during aging and development of osteoarthritis (OA), using a rabbit model of induced OA. METHODS: Six mature and 6 aged rabbits underwent anterior cruciate ligament transection and were killed 4 and 8 weeks after surgery, respectively, to create early-grade and advanced-grade OA. RNA from articular cartilage and menisci was examined for expression of the genes caspase 8, Fas, Fas ligand, p53, aggrecanase, matrix metalloproteinase 1 (MMP-1), and MMP-3. A second cohort of animals that had undergone no intervention in the joint was also killed. Parametric data were analyzed with analysis of variance and Student's t-tests, while nonparametric data were assessed with the Mann-Whitney U test. RESULTS: Expression levels of Fas, caspase 8, FasL, and MMP-1 were significantly higher (>100%) in aged cartilage compared with mature cartilage (P < 0.05). After induction of OA, expression of apoptotic genes in aged rabbits remained high, while significant up-regulation of Fas and caspase 8 (nearly 150% increase) was observed in mature rabbits (P < 0.05). No significant up-regulation of these genes was observed in the menisci of aged or mature rabbits prior to or after induction of OA. Development of OA occurred more rapidly in aged cartilage compared with mature cartilage (P < 0.05). CONCLUSION: Differential expression of apoptotic and matrix-degrading genes occurs in aged compared with mature cartilage, both at baseline and during development of OA. This may be responsible for faster degradation of aged cartilage and its predisposition for developing OA.

Characterization of mature vs aged rabbit articular cartilage: analysis of cell density, apoptosis-related gene expression and mechanisms controlling chondrocyte apoptosis.

OBJECTIVE: The prevalence of osteoarthritis (OA) is increased in aged individuals and a direct correlation between chondrocyte apoptosis and cartilage degradation secondary to OA has been demonstrated. To address the question of whether age predisposes articular cartilage to apoptosis, the objective of the present study was to characterize and compare in aged and mature non-OA rabbit articular cartilage, cell density and expression levels of specific genes associated with apoptosis. Mechanistic studies on the inhibition of induced apoptosis were also carried out. METHODS: Grade I (non-OA) femoral condyles and tibial plateaus from mature and aged rabbits were taken for assessment of viable cell density (VCD) and mRNA (reverse transcription-polymerase chain reaction) expression levels of the pro-apoptotic genes, Fas, Fas ligand (FasL), caspase-8, inducible nitric oxide synthase (iNOS) and p53. In vitro insulin-like growth factor (IGF-1)-mediated inhibition of nitric oxide (NO)-induced apoptosis was also examined using sodium nitroprusside (SNP) as NO donor. RESULTS: VCD was decreased 50-70% in aged articular cartilage relative to mature cartilage. mRNA expression levels of Fas, FasL, caspase-8 and p53 were higher in aged cartilage than in mature cartilage. iNOS expression was unchanged. IGF-1-mediated inhibition of NO-induced apoptosis was dose-dependent and reversed with addition of phosphatidylinositol-3 kinase inhibitor. CONCLUSIONS: This controlled animal model study demonstrates that age predisposes articular cartilage to changes in VCD and expression levels of specific pro-apoptotic genes. It is significant that these findings were demonstrated on cartilage that showed no prior signs of OA; it is also possible that such changes are a prelude to the age-related development of OA.