Effect of recombinant human platelet-derived growth factor-BB-coated sutures on Achilles tendon healing in a rat model: A histological and biomechanical study.

PURPOSE: Repairing tendon injuries with recombinant human platelet-derived growth factor-BB has potential for improving surgical outcomes. Augmentation of sutures, a critical component of surgical tendon repair, by coating with growth factors may provide a clinically useful therapeutic device for improving tendon repair. Therefore, the purpose of this study was to (a) coat Vicryl sutures with a defined dose of recombinant human platelet-derived growth factor-BB without additional coating excipients (e.g. gelatin), (b) quantify the recombinant human platelet-derived growth factor-BB released from the suture, and (c) use the recombinant human platelet-derived growth factor-BB-coated sutures to enhance tendon repair in a rat Achilles tendon transection model. METHODS: Vicryl sutures were coated with 0, 0.3, 1.0, and 10.0 mg/mL concentrations of recombinant human platelet-derived growth factor-BB using a dip-coating process. In vitro release was quantified by an enzyme-linked immunosorbent assay. Acutely transected rat Achilles tendons were repaired using one of the four suture groups (n = 12 per group). Four weeks following repair, the tensile biomechanical and histological (i.e. collagen organization and angiogenesis) properties were determined. RESULTS: A dose-dependent bolus release of recombinant human platelet-derived growth factor-BB occurred within the first hour in vitro, followed by a gradual release over 48 h. There was a significant increase in ultimate tensile strength (p < 0.01) in the two highest recombinant human platelet-derived growth factor-BB dose groups (1.9 ± 0.5 and 2.1 ± 0.5 MPa) relative to controls (1.0 ± 0.2 MPa). The modulus significantly increased (p = 0.031) with the highest recombinant human platelet-derived growth factor-BB dose group (7.2 ± 3.8 MPa) relative to all other groups (control: 3.5 ± 0.9 MPa). No significant differences were identified for the maximum load or stiffness. The histological collagen and angiogenesis scores were comparable in all groups, although there was a trend for improved collagen organization in the recombinant human platelet-derived growth factor-BB-treated groups (p = 0.054). CONCLUSIONS: The results of this study suggest that recombinant human platelet-derived growth factor-BB can be used to reproducibly coat Vicryl sutures and improve remodeling in a rat Achilles tendon transection model by significantly decreasing the resulting cross-sectional area, thus improving the material properties of the repaired tendon.

Ultrasonic assessment of extracellular matrix content in healing Achilles tendon.

Although several imaging modalities have been utilized to observe tendons, assessing injured tendons by tracking the healing response over time with ultrasound is a desirable method which is yet to be realized. This study examines the use of ultrasound for non-invasive monitoring of the healing process of Achilles tendons after surgical transection. The overall extracellular matrix content of the transection site is monitored and quantified as a function of time. B-mode images (built from successive A-scan signatures) of the injury site were obtained and compared to biomechanical properties. A quantitative measure of tendon healing using the extracellular matrix (ECM) content of the injury site was analyzed using linear regression with all biomechanical measures. Contralateral tendons were used as controls. The trend in the degree of ECM regrowth in the 4 weeks following complete transection of excised tendons was found to be most closely paralleled with that of linear stiffness (R(2) = 0.987, p < .05) obtained with post-ultrasound biomechanical tests. Results suggest that ultrasound can be an effective imaging technique in assessing the degree of tendon healing, and can be used to correlate structural properties of Achilles tendons.

Tendon repair augmented with a novel circulating stem cell population.

Tendon ruptures are common sports-related injuries that are often treated surgically by the use of sutures followed by immobilization. However, tendon repair by standard technique is associated with long healing time and often suboptimal repair. Methods to enhance tendon repair time as well as the quality of repair are currently unmet clinical needs. Our hypothesis is that the introduction of a unique stem cell population at the site of tendon transection would result in an improved rate and quality of repair. Achilles tendons of fifty-one Sprague-Dawley rats were transected and suture-repaired. In half of the rats, a biodegradable scaffold seeded with allogenic circulating stem cells was placed as an onlay to the defect site in addition to the suture repair. The other half was treated with suture alone to serve as the control group. Animals were randomized to a two-, four-, or six-week time group. At the time of necropsy, tendons were harvested and prepared for either biomechanical or histological analysis. Histological slides were evaluated in a blinded fashion with the use of a grading scale. By two weeks, the experimental group demonstrated a significant improvement in repair compared to controls with no failures. Average histological scores of 0.6 and 2.6 were observed for the experimental and control group respectively. The experimental group demonstrated complete bridging of the transection site with parallel collagen fiber arrangement. By four weeks, both groups showed a continuing trend of healing, with the scaffold group exceeding the histological quality of the tissue repaired with suture alone. Biomechanically, the experimental group had a decreasing cross-sectional area with time which was also associated with a significant increase in the ultimate tensile strength of the tendons, reaching 4.2MPa by six weeks. The experimental group also achieved a significantly higher elastic toughness by six weeks and saw an increase in the tensile modulus, reaching 31Mpa by six weeks. The use of circulating stem cells as an adjunct in tendon repair demonstrates superior biomechanical properties and an improved level of histological organization, when compared to the suture alone control group. These improvements were not previously observed when gene therapy, protein therapy, or current tissue engineering technologies were used.

Tendon phenotype should dictate tissue engineering modality in tendon repair: a review.

Advancements in the technical aspects of tendon repair have significantly improved the treatment of tendon injuries. Arthroscopic techniques, suture material, and improved rehabilitation have all been contributing factors. Biological augmentation and tissue engineering appear to have the potential to improve clinical outcomes as well. After review of the physiology of tendon repair, three critical components of tissue engineering can be discerned: the cellular component, the carrier vehicle (matrix or scaffold), and the bioactive component (growth factors, platelet rich plasma). These three components are discussed with regard to each of three tendon types: Intra-synovial (flexor tendon), extra-synovial (Achilles tendon), and extra-synovial tendon under compression (rotator cuff). Scaffolds, biologically enhanced scaffolds, growth factors, platelet rich plasma, gene therapy, mesenchymal stem cells, and local environment factors in combination or alone may contribute to tendon healing. In the future it may be beneficial to differentiate these modes of healing augmentation with regard to tendon subtype.

In vitro analysis of an rhGDF-5 suture coating process and the effects of rhGDF-5 on rat tendon fibroblasts.

We describe a dipcoating method of coating 4-0 VICRYL sutures with recombinant human growth and differentiation factor-5 (rhGDF-5), and analyze the in vitro effects of rhGDF-5 on rat tendon fibroblasts (RTFs). Part I: Eight 4-0 VICRYL sutures were coated in a dipcoat solution using rhGDF-5 solutions at concentrations of 0, 40, 200, and 1000 µg/ml (n=32). ELISA was performed to determine the amount of rhGDF-5 that was transferred on the suture. Part II: Using a dipcoat solution of 200 µg/ml, four sutures were passed through rat tendon, and quantified ELISA was again performed. Cell proliferation, collagen synthesis, and RTF cell migration were also analyzed. The differences in the amount rhGDF-5 transferred on the suture between the different concentration groups were statistically significant. Furthermore, rhGDF-5-stimulated RTF cell migration, cell proliferation, and collagen synthesis at dipcoat concentrations of rhGDF-5 of at least 200 µg/ml.

Augmentation of zone II flexor tendon repair using growth differentiation factor 5 in a rabbit model.

PURPOSE: Modulation of zone II flexor tendon repair healing using growth factors may reduce the incidence of complications, such as rupture and fibrosis. We hypothesized that sutures coated with growth differentiation factor 5 (GDF5) will stimulate the healing of zone II flexor tendon repairs. METHODS: We created and immediately repaired zone II flexor tendon lacerations in the second and fourth toe of the right forepaw of 44 New Zealand White rabbits. One tendon was repaired with suture coated with GDF5, whereas the other tendon was repaired with suture without GDF5 (control). We randomized the allocation of GDF5 and control suture to either toe. A proximal tenotomy of the flexor digitorum profundus at the level of the wrist was performed to relieve tension on the more distal repairs. Rabbits were euthanized at 21 or 42 days after repair. Four rabbits (8 tendons) underwent histological analysis at each time point; the remaining repairs were tested biomechanically in a blinded fashion. RESULTS: Control tendons demonstrated distinct borders at the transection site and less endogenous repair at 3 weeks. The Soslowsky histological score for collagen was better in the GDF5 group at both time points (p≤.003). All tendons failed at the repair site. The maximum load was significantly greater (p=.04) in the GDF5 group (11.6 ± 3.5 N) compared with control tendons (8.6 ± 3.0 N) at 3 weeks. The maximum load was not significantly different (p=.12) at 6 weeks. We observed no significant differences in stiffness at either time point (p>.11). CONCLUSIONS: The results demonstrate that GDF5 has an early beneficial effect on tendon healing in zone II flexor tendon repairs in a rabbit flexor tendon injury model.

Histologic stages of healing correlate with restoration of tensile strength in a model of experimental tendon repair.

Much current research is focused on biologic enhancement of the tendon repair process. To evaluate the different methods, which include a variety of gene therapy and tissue engineering techniques, histological and biomechanical testing is often employed. Both modalities offer information on the progress and quality of repair; however, they have been historically considered as two separate entities. Histological evaluation is a less costly undertaking; however, there is no validated scoring scale to compare the results of different studies or even the results within a given study. Biomechanical testing can provide validated outcome measures; however, it is associated with increased cost and is more labor intensive. We hypothesized that a properly developed, objective histological scoring system would provide a validated outcome measure to compare histological results and correlate with biomechanics. In an Achilles tendon model, we have developed a histological scoring scale to assess tendon repair. The system grades collagen orientation, angiogenesis, and cartilage induction. In this study, histology scores were plotted against biomechanical testing results of healing tendons which indicated that a strong linear correlation exists between the histological properties of repaired tendons and their biomechanical characteristics. Concordantly, this study provides a pragmatic and financially feasible means of evaluating repair while accounting for both the histology and biomechanical properties observed in surgically repaired, healing tendon.

The effect of growth differentiation factor-5-coated sutures on tendon repair in a rat model.

Tendon ruptures are common injuries that are often treated surgically. Growth Differentiation Factor-5 (GDF-5) has been shown to accelerate tendon healing with varying degrees of success. We used a novel technique to apply recombinant human GDF-5 (rhGDF-5) to suture and hypothesized that controlled, local delivery of rhGDF-5 can be used to enhance tendon repair. Tendons of 92 rats were transected and repaired with sutures. All researchers were blinded to the following treatment groups (24 rats in each group): 0 rhGDF (control), 24 ng/cm rhGDF, 55 ng/cm rhGDF, and 556 ng/cm rhGDF. Rats were euthanized at 3 weeks (n = 48) and at 6 weeks (n = 48). Sutures were coated with rhGDF-5 using a novel dip-coat technique. Enzyme-linked immunosorbent assay confirmed consistent and reproducible delivery of rhGDF-5. Within each group, 8 were tested biomechanically, and 4 were assessed histologically. Histologic grading at 3 weeks showed improved healing in tendons repaired with coated suture versus controls. By 6 weeks, there were no significant differences. At 3 weeks, minimal isolated cartilage formation was observed; 6-week samples showed more extensive presence, typically surrounding suture fibers. At 3 weeks, tendons repaired with rhGDF-5-coated sutures resulted in significantly higher ultimate tensile load and stiffness compared with control sutures (P < .05) At 6 weeks, there were no significant differences in the mechanical properties of repaired tendons. At 3 weeks, rhGDF-5 induced significant tendon hypertrophy that was more pronounced than at 6 weeks. In addition, tendons repaired with rhGDF-5 showed an increased rate of healing versus control repairs at 3 weeks. This study showed that a novel dip-coating technique can be used to deliver growth factors in varying concentrations to local repair sites to accelerate tendon healing.

Gene therapy and tissue engineering in repair of the musculoskeletal system.

Historically, surgeons have sought and used different procedures in order to augment the repair of various skeletal tissues. Now, with the completion of the Human Genome Project, many researchers have turned to gene therapy as a means to aid various ailments. In the orthopedic field, many strides have been made toward using gene therapy and tissue engineering in a clinical setting. In this review, several studies are outlined in different areas that gene therapy has or will influence orthopedic surgery. Gene therapy and tissue engineering can aid in fracture healing and spinal fusions by inducing bone formation, ligamentous repairs by increasing the production of connective tissue fibers, intervertebral disc disease by creating potential replacements, and articular cartilage repairs by providing means to improve cartilage. As we continue to see great contributions, such as the few mentioned here, this field will continue to mature and develop.

Suppression of nuclear factor-kappa B activity by nitric oxide and hyperoxia in oxygen-resistant cells.

Inhaled nitric oxide (iNO) is used clinically to treat pulmonary hypertension in newborns, often in conjunction with hyperoxia (NO/O2). Prolonged exposure to NO/O2 causes synergistic lung injury and death of lung epithelial cells. To explore the mechanisms involved, oxygen-resistant HeLa-80 cells were exposed to NO +/- O2. Exposure to NO and O2 induced a synergistic cytotoxicity, accompanied with apoptotic characteristics, including elevated caspase-3-like activity, Annexin V incorporation, and nuclear condensation. This apoptosis was associated with a synergistic suppression of NF-kappaB activity. Cells lacking functional NF-kappaB p65 subunit were more sensitive to NO/O2 than their wild type counterparts. This injury was partially rescued by transfection with a p65 expression construct, suggesting an inverse relationship between NF-kappaB and susceptibility to the cytotoxicity of NO/O2. Despite the reduced NF-kappaB activity in cells exposed to NO +/- O2, IkappaBalpha was degraded, suggesting that pathways regulating the steady-state levels of IkappaB were not involved. However, exposure to NO/O2 caused a marked reduction in nuclear localization and an increase in protein carbonyl formation of NF-kappaB p65 subunit. These results suggest that NO/O2-induced apoptosis occurs by suppressing NF-kappaB activity.