Frederick L. Harwood

The Effect of Immobilization on Collagen Turnover in Connective Tissue: A Biochemical-Biomechanical Correlation

Immobilization of the knee joint for 9 weeks results in a reduction of the mechanical properties in the lateral collateral ligament. Specifically, ligament stiffness is reduced in this tissue. No statistical change in collagen mass was detected for the medial collateral ligament (MCL) or patellar tendon. An increase in collagen turnover (synthesis and degradation) was, however, found in the immobilized medial collateral ligament and patellar tendon. It is thus proposed that stiffness reduction is due to a change in the ligament substance itself, rather than a result of tissue atrophy.

Collagen Cross-Linking Alterations in Joint Contractures: Changes in the Reducible Cross-Links in Periarticular Connective Tissue Collagen After Nine Weeks of Immobilization

A significant increase in the NaBH4 reducible intermolecular cross-links in the 9 week immobilized rabbit periarticular connective tissue was found. Dihydroxylysinonorleucine, hydroxylysinonorleucine, and histidinohydroxymerodesmosine were the major cross-links which increased during the period of immobilization. No change in the hydroxlysine/lysine ratio between the immobilized and control periarticular connective tissue collagen was detected during the 9 weeks of enforced immobilization of the rabbit knee. Since the collagen mass in the immobilized periarticular connective tissue does not change, or at most is reduced 10%, we suggest that there is an increase in collagen cross-links expressed both in per unit weight of collagen and on the basis of collagen mass per knee due to the lack of physical stress and motion.

The effects of hyaluronan during the development of osteoarthritis

Ninety-nine mature New Zealand White (NZW) rabbits underwent unilateral anterior cruciate ligament transection (ACLT) and were divided into three groups. The contralateral non-operated knees served as controls. The first group (SA) received intra-articular injections of 0.3 ml hyaluronan (HA: MW; 8 x 10(5)) beginning 4 weeks after ACLT, once a week for 5 weeks. The second group (SV) was injected with vehicle (carrier of HA) in the same fashion as the SA group. The third group (SN) served as a nontreatment group post ACLT. All animals were killed 9 weeks post-surgery and were assessed by gross morphology, histomorphometry and biochemical analysis. Gross morphologic changes on the femoral cartilage in the SA group were less severe than those in the SV and SN groups. Cartilage thickness, cartilage area, and thickness of synovial lining cell layer histomorphometric parameters were measured, showing a positive effect of HA on the preservation of articular cartilage and synovial tissue. Similarly, the cartilage and synovial tissues from knees injected with HA did not demonstrate significant alterations from contralateral controls as measured by biochemical analysis [i.e., water content, pyridinoline concentration, glycosaminoglycan (GAG) content for the cartilage, and DNA concentration for the synovial tissue].

The effects of increased tension on healing medial collateral ligaments

The effects of motion and increased levels of stress on the biomechanical, biochemical, and morphological properties of healing medial collateral ligaments were assessed in a rabbit model. In one group, the medial collateral ligament of the left hindlimb was transected and allowed to heal with cage activity for either 6 or 12 weeks. In another group, the transected ligaments were permitted to heal for 4 weeks and then were placed under increased stress by inserting a stainless steel pin perpendicularly underneath the healing medial collateral ligament. The animals were allowed cage activity for an additional 2 or 8 weeks. The varus-valgus joint laxity and the stress-strain properties of the medial collateral ligament substance were obtained. Further, the quan tity of total collagen, amount and ratio of the collagen cross-links, dihydroxylysinonorleucine and hydroxyly sinonorleucine, and the histologic appearance of the healing medial collateral ligaments were evaluated for all groups. At 6 weeks, knees with a transected medial collateral ligament were twice as lax as the controls. However, joints with the stainless steel tension pin had varus-valgus values approximately 1.5 times those of the controls. At 12 weeks, joints with increased stress were not statistically different from the controls. The group that had healing with increased stress for 12 weeks produced the highest stress for a given strain compared to any other group. Also, the total collagen levels and the ratio of dihydroxylysinonorleucine/hy droxylysinonorleucine were the closest to normal of any transected group. Finally, qualitative histologic improve ments were seen, including a more longitudinal arrange ment of collagen fibers and decreased cellularity.

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 tendons 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.

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.

The chondrogenesis of rib perichondrial grafts for repair of full thickness articular cartilage defects in a rabbit model: A one year postoperative assessment

The purpose of this study was to investigate the results or rib perichondrial grafting after the creation of a full thickness articular cartilage defect. In a rabbit model, rib perichondrium was used to repair defects created in the femoral condyle. The formation of repair tissue (neocartilage) and its chondrogenesis into a tissue resembling articular cartilage was found over time. The gross, histological and biochemical characteristics of the neocartilage were evaluated at intervals of 6, 12, 18, 26 and 52 weeks post transplant, and compared to normal articular cartilage. The neocartilage was characterized by the early formation of relatively large amounts of glycosaminoglycans. A steady increase in the proportion of type II collagen over the time periods was also observed. Improved attachment of the neocartilage to host tissues was seen over the period of 6 to 52 weeks. Successful grafts were seen to proliferate to fill the articular defect and to undergo a chondrogenesis over a post transplant time period of one year.

Donor cell fate in tissue engineering for articular cartilage repair.

Articular cartilage repair is a clinical challenge because of its limited intrinsic healing potential. Considerable research has focused on tissue engineering and transplantation of viable chondrogenic cells to enhance cartilage regeneration. However, the question remains: do transplanted allogenic cells survive in the repair with time? This study assessed donor cell fate after transplantation of male New Zealand White rabbit perichondrium cell and polylactic acid constructs into osteochondral defects created in the medial femoral condyles of female New Zealand White rabbits. Repair tissue was harvested at 0, 1, 2, 3, 7, and 28 days after implantation and was evaluated for cell viability and total cell number using confocal microscopic analysis. The number of donor cells in each sample was estimated using quantitative polymerase chain reaction targeting a gender-specific gene present on the Y-chromosome, the sex-determining region Y gene, and a control deoxyribonucleic acid present in male and female cell deoxyribonucleic acid, the matrix metalloproteinase-1 gene promoter. Average cell viability was found to be 87% or more at all times. Donor cells were present in repair tissue for 28 days after implantation. However, the number of donor cells declined from approximately 1 million at Time 0 to approximately 140,000 at 28 days. This decline in donor cells was accompanied by a significant influx of host cells into the repair tissue. This study shows that the sex-determining region Y gene is a valuable marker for tracking the fate of transplanted allogenic cells in tissue engineering.

The Effects of Hyaluronan on Tissue Healing After Meniscus Injury and Repair in a Rabbit Model

To assess the effect of hyaluronan on meniscus injury and repair, we had 35 mature New Zealand White rabbits undergo bilateral meniscus injury and repair (19 in the peripheral region, and 16 in the inner region). A longitudinal tear was created in the medial meniscus and repaired with horizontally placed nylon sutures. The left knee joint received intraarticular injections of hyaluronan 1 week after surgery and once a week for 5 weeks. The right knees were injected with phosphate-buffered saline (the carrier vehicle of the hyaluronan). Twelve weeks after repair, tears in the peripheral region showed gross and histologic evidence of healing, with no difference between the vehicle- and hyaluronan-treated menisci. Biochemically, the ratio of reducible collagen cross-links in the hyaluronan-treated menisci was significantly higher than in the vehicle-treated menisci, indicating greater level of collagen remodeling. Biomechanically the vehicle- and hyaluronan-treated menisci demonstrated similarly high tearing load and fracture toughness. In the inner region, poor healing response was observed grossly and histologically in both treatment groups. Water content in the hyaluronan-treated menisci was significantly lower than in the vehicle-treated menisci, indicating a lower level of swelling. Hyaluronan treatment stimulated collagen remodeling in the peripheral region and inhibited swelling of the meniscus repaired in the inner region.