The Effects Of Trypsinization And Mineralization On Intrasynovial Tendon Allograft Healing To Bone

Chapter one:A comparison of anterior cruciate ligament, flexor digitorum profundus, Achilles tendon and patellar tendon:an in vitro mechanical and histological study in dogsPurpose:Intrasynovial tendon region, the tendon surface is covered by epitenon, which includes several layers of epitenon cells seeding on the surface. extrasynovial tendon is wrapped by loose connective tissue or paratenon. To compare the differences among anterior cruciate ligament (ACL), flexor digitorum profundus (FDP), Achilles tendon (N=20) and patellar tendon (PAT) with mechanical and histological methods.Methods:20hind legs were used to harvest anterior cruciate ligament (N=20), flexor digitorum profundus, Achilles tendon (N=20) and patellar tendon (N=20). These samples were evaluated with friction testing, indentation testing and histology.Results:Based on the friction testing, in the first cycle, compared with patellar tendon (336.62±110.87mN) and Achilles tendon (147.89±54.73mN), flexor digitorum profundus (47.24±14.40mN) and anterior cruciate ligament (51.98±12.10mN) showed significantly lower friction force. In the fifth cycle, compared with patellar tendon (414.29±104.49mN) and Achilles tendon (260.11±99.11mN), flexor digitorum profundus (88.73±18.33mN) and anterior cruciate ligament (126.88±50.83mN) showed significantly lower friction force. In the tenth cycle, compared with patellar tendon (442.12±106.69mN), flexor digitorum profundus (132.53±26.08mN), anterior cruciate ligament (196.69±84.80mN) and Achilles tendon (311.89±124.78mN) showed significantly lower friction force. After100cycles, compared with patellar tendon (571.06±132.50mN), flexor digitorum profundus (397.26±39.98mN) and anterior cruciate ligament (396.53±90.59mN) showed significantly lower friction force. Based on the indentation testing, compared with patellar tendon (2.85±0.43MPa), flexor digitorum profundus (4.11±0.34MPa), anterior cruciate ligament (3.54±0.32MPa) and Achilles tendon (4.71±0.38MPa) showed significantly higher compressive moduli (P＜0.05). Histological results showed that flexor digitorum profundus and anterior cruciate ligament owned a very smooth surface, with a single layer of epitenon cells covering. While patellar tendon and Achilles tendon owned a rough surface and a layer of paratenon. Lubricin was found on the surface and extracellular matrix of the flexor digitorum profundus and anterior cruciate ligament. There was only very limited lubricin expression on the surface and extracellular matrix of patellar tendon and Achilles tendon. Conclusion:Both flexor digitorum profundus and anterior cruciate ligament showed significantly lower friction force compared with patellar tendon and Achilles tendon. In comparision with patellar tendon and Achilles tendon, more lubricin was found on the surface and extracellular matrix of the flexor digitorum profundus and anterior cruciate ligament. This is consistent with the friction results.Chapter two:Optimization of intrasynovial tendon mineralzation process: an in vitro studyPurpose:Tendon-to-bone integration is a great challenge for tendon or ligament reconstruction regardless of use of autograft or allograft tendons. The purpose of the current study was to mineralize the tendon, thus transforming the tendon-to-bone into a "bone-to-bone" interface for healing.Methods:Sixty dog flexor digitorum profundus (FDP) tendons were divided randomly into5groups as follows:1) normal FDP tendon,2) CaP (Non-extraction and mineralization without fetuin),3) CaPEXT (Extraction by Na2HPO4and mineralization without fetuin),4) CaPFetuin (Non-extraction and mineralization with fetuin), and5) CaPEXTFetuin (Extraction and mineralization with fetuin). The tendon segments in two extration groups were suspended in3%Na2HPO4solution for3days. The mineralization solution for two extration groups contained fetuin. These samples were evaluated by histology, scanning electron microscopy and biomechanical testing.Results:The calcium and phosphate content significantly increased in tendons treated with combination of extraction and fetuin compared to the other treatments. Histology also revealed a dense mineral deposition throughout the tendon outer layers and penetrated into the tendon to a depth of200μm in a graded manner. Compressive moduli were significantly lower in the four mineralized groups compared with normal control group. No significant differences in maximum failure strength or stiffness were found in the suture pull-out test among all groups.Conclusion:Mineralization of tendon alters the interface from tendon to bone into mineralized tendon to bone, which may facilitate tendon-to-bone junction healing following tendon or ligament reconstruction.Chapter three:The effects of trypsinization and mineralization on intrasynovial tendon allograft healing to bone:an in vivo rabbit modelPurpose:the purpose of the current study was to develop a novel technology to enhance tendon to bone conjunction healing by mineralizing and trypsinizing an intrasynovial tendon allograft using a rabbit tibia bone tunnel model.Methods:8rabbit flexor digitorum profundus (FDP) tendons were used to optimize the duration of trypsinization process. Other24FDP tendons were divided into two groups:Control group (n=8) and Trypsinization and mineralization (TM) group (n=8).4tendons in each group were used for in vitro evaluations of trypsinization and mineralization.8tendons in each group were transplanted into the proximal tibial bone tunnels in rabbits. The results were evaluated with histology and mechanical testing.Results:10minutes was found to be the best duration of trypsinization. The mineral deposition was visible inside the tendon in a graded manner. No mineral was deposited in the un-mineralized portion of the tendon. Only very limited staining of lubricin was found on the extracellular matrix of the trypsinized and mineralized tendon, and no staining was found on the tendon surface. There was no significant differences in maximum failure strength and linear stiffness between normal group and TM group. There was a thin fibrous scar tissue band formed at the graft-to-bone interface in the control group. However, obvious new bone formation was observed only inside the tendon graft in the TM group. There was visible fibrocartilage layer formed at the bone tunnel entrance in the TM group.Conclusion:the current study for the first time explored the effects of trypsinization and mineralization on the intrasynovial allograft healing to bone tunnel. Trypsinization and mineralization showed some beneficial effects on the chondrogenesis and osteogenesis, as well as integration of intrasynovial tendon graft, although mechanical strength were the same between two groups in this short-term in vivo study.