Objectives

The lack of effective treatment for cartilage defects has prompted investigations using tissue engineering techniques for their regeneration and repair. The success of tissue-engineered repair of cartilage may depend on the rapid and efficient adhesion of transplanted cells to a scaffold. Our aim in this study was to repair full-thickness defects in articular cartilage in the weight-bearing area of a porcine model, and to investigate whether the CD44 monoclonal antibody biotin-avidin (CBA) binding technique could provide satisfactory tissue-engineered cartilage.

Introduction and Aims: Although skeletal muscles have remarkable potential for adaptation, the amount of muscle length increase during gradual limb lengthening is always less than the amount of bone lengthening. The purpose of this study was to analyse gene expression in skeletal muscle undergoing adaptation to limb lengthening. Method: Ten adult goats were randomly divided into two groups of five animals. Group 1 underwent 20% (43–47mm) standard Ilizarov tibial lengthening and group 2 served as un-operated control. Muscle tissues from proximal myotendenous junctions of Peroneus Longus were harvested from the lengthened limb in the distraction group and corresponding limb in the control group and immediately snap frozen in liquid nitrogen. To screen for genes potentially associated with sarcomerogenesis, microarray technology was employed. Biotin labeled cRNA was hybridised to Affymetrix HU133A GeneChips, containing 22,284 gene transcripts. All created data files were analysed using computer software GeneSpring 5.0. Results: In both groups, 5092 (23%) gene transcripts flagged present. Thirty-two of these transcripts were differentially expressed between distracted and control groups (p < 0.05). Represented by these transcripts were 12 known and three unknown genes, which were up-regulated in lengthened muscles by more than 2.0 fold. The substantially up-regulated genes identified were MYOZ2 (myozenin 2), MYL4 (embryonic myosin alkali light chain), MYL6 (myosin light polypeptide 6), CRYAB (crystalline), PFN2 (profiling 2), ARPP-19 (cyclic AMP phosphorprotein), TUBB2 (tubulin beta 2), PPP1R12 (protein phosphatase 1), RCOR (REST corepressor), LIM (LIM protein), FN1 (fibronectin 1), ACTC (alpha-actin), and hypothetical protein FLJ10111. Among the genes found to be up-regulated are genes involved in the myogenesis pathway. Myozenin 2 gene is associated with the signalling and activity of Calcineurin/Calsarcin that plays a significant role in muscle cell proliferation and myofiber type differentiation. Crystallin gene may be involved in promoting muscle survival during differentiation. The functionality of the remaining genes range from cytoskeletal organisation (TUBB2), cyto-skeletal structure (PFN2, MYL4, MYL6), cell adhesion and motility (FN1), muscle development and differentiation (FHL1 and LIM), intercellular adhesion and intermediate filament organisation (PNN), muscle contraction and relaxation (PPP1R12A), neuronal-specific gene silencing (RCOR), and PKA-dependent intracellular messaging (ARPP-19). Conclusion: The findings suggest that tension stress observed during gradual limb lengthening using standard Ilizarov distraction protocol activates expression of genes involved in skeletal muscle growth, differentiation, and neogenesis. On-going studies involving immunohistochemistry, RT-PCR, and in situ hybridisation to confirm cellular localisation of up-regulated genes are underway

Introduction: A number of clinical and experimental studies suggest that an intact nervous system is essential for normal fracture healing. In the present study, we analysed the occurrence of regenerating and mature nerve fibres over time in fracture callus. Using antibodies against neuronal proteins specific for nerve regeneration (growth associated protein – GAP-43) and nerve maturity (protein gene product – PGP 9.5) it is possible to demonstrate regeneration and end differentiation of nerves by immunohistochemistry. Methods: Twelve male Sprague Dawley rats, weighing 230–290 g were used. The right tibias were fractured under HypnormÒ anaesthesia and fixed with a 17-G cannula needle in the medullary canal. The left un-fractured tibia served as an internal control. X-rays was used to monitor progress of fracture healing. Three rats were killed at 3 days, 1, 2 and 3 weeks post-fracture and right and left tibia were prepared for immunohistochemistry. The tissue sections (15 mm thick) were incubated with antiserum to GAP-43 and then with biotinylated antibodies. Cy2-conjugated avidin was used for the fluorescent staining. For double staining, after the staining with first antibody, the sections were incubated with avidin blocking solution followed by biotin blocking solution. Incubation with the second antiserum to PGP 9.5 was performed in the same manner as for the first peptide. For fluorescent staining of PGP 9.5, the sections were incubated with Cy3-conjugated avidin. A Nikon epifluorescence microscope was used for photog. Results: In the un-fractured tibia. PGP 9.5-positive nerve fibres were consistently identified in periosteum, muscles and connective tissues. A number of nerve fibres also expressed GAP-43, although there were no signs of nerve sprouting, i.e. regeneration. In the fractured tibia, many GAP-43-positive nerves were identified already at 3 days post-fracture in the hematoma and periosteum. At 1 week, abundant sprouting of these nerves was seen in cartilaginous callus and hyperplastic periosteum. A number of nerve terminals were observed very close to the chondroid cells in the fibrocartilage of the fracture gap. At 2 and 3 weeks, GAP 43-positive fibres gradually shifted from the fibrocartilage area towards the outlying hyperplastic periosteum. Double staining studies showed that an increased expression of GAP-43 as compared to PGP 9.5 occurred in the early period of fracture healing. This relationship changed at 3 weeks when enhanced PGP 9.5 and less GAP 43 expression was found. Discussion: Our study suggests that there was an intense nerve regeneration in the early phase of fracture healing. Thus, a prominent expression of GAP-43 was seen in sprouting nerves in the hyperplastic periosteum and the callus fibrocartilage as early as 1 week post-fracture. This expression remained high in the fractures up to 3 weeks, when healing was essentially completed. Possibly, this persistent occurrence of GAP-43 is necessary for the ensuing ossification and bone remodeling. PGP 9.5 expression was markedly low at one week, but became pronounced at 3 weeks, probably reflecting functional maturation of the regenerated nerve in the healing fracture. It may prove that strong regenerative capability of nerves seen in the fractures is a prerequisite for normal fracture healing. Our results point to the possibility that regenerating nerves provide the delivery system for GAP-43 and other neuronal mediators required for normal callus formation and/or neovascularization

Objectives

The objective of this study was to develop a test for the rapid (within 25 minutes) intraoperative detection of bacteria from synovial fluid to diagnose periprosthetic joint infection (PJI).