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. Methods. Cartilage defects were created in the load-bearing region of the lateral femoral condyle of mini-type pigs. The defects were repaired with traditional tissue-engineered cartilage, tissue-engineered cartilage constructed with the biotin-avidin (BA) technique, tissue-engineered cartilage constructed with the CBA technique and with autologous cartilage. The biomechanical properties, Western blot assay, histological findings and immunohistochemical staining were explored. Results. The CBA group showed similar results to the autologous group in biomechanical properties, Moran’s criteria, histological tests and Wakitani histological scoring. Conclusions. These results suggest that tissue-engineered cartilage constructed using the CBA technique could be used effectively to repair cartilage defects in the weight-bearing area of joints. Cite this article: H. Lin, J. Zhou, L. Cao, H. R. Wang, J. Dong, Z. R. Chen. Tissue-engineered cartilage constructed by a biotin-conjugated anti-CD44 avidin binding technique for the repairing of cartilage defects in the weight-bearing area of knee joints in pigs. Bone Joint Res 2017;6:–295. DOI: 10.1302/2046-3758.65.BJR-2016-0277

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

A rat model of lumbar root constriction with an additional sympathectomy in some animals was used to assess whether the sympathetic nerves influenced radicular pain. Behavioural tests were undertaken before and after the operation. On the 28th post-operative day, both dorsal root ganglia and the spinal roots of L4 and L5 were removed, frozen and sectioned on a cryostat (8 μm to 10 μm). Immunostaining was then performed with antibodies to tyrosine hydroxylase (TH) according to the Avidin Biotin Complex method. In order to quantify the presence of sympathetic nerve fibres, we counted TH-immunoreactive fibres in the dorsal root ganglia using a light microscope equipped with a micrometer graticule (10 x 10 squares, 500 mm x 500 mm). We counted the squares of the graticule which contained TH-immunoreactive fibres for each of five randomly-selected sections of the dorsal root ganglia. The root constriction group showed mechanical allodynia and thermal hyperalgesia. In this group, TH-immunoreactive fibres were abundant in the ipsilateral dorsal root ganglia at L5 and L4 compared with the opposite side. In the sympathectomy group, mechanical hypersensitivity was attenuated significantly. We consider that the sympathetic nervous system plays an important role in the generation of radicular pain

Aims

This study aimed to demonstrate the promoting effect of elastic fixation on fracture, and further explore its mechanism at the gene and protein expression levels.

Objectives

The role of mechanical stress and transforming growth factor beta 1 (TGF-β1) is important in the initiation and progression of osteoarthritis (OA). However, the underlying molecular mechanisms are not clearly known.

The aim of this study was to investigate the occurrence of tissue hypoxia and apoptosis at different stages of tendinopathy and tears of the rotator cuff.

We studied tissue from 24 patients with eight graded stages of either impingement (mild, moderate and severe) or tears of the rotator cuff (partial, small, medium, large and massive) and three controls. Biopsies were analysed using three immunohistochemical techniques, namely antibodies against HIF-1α (a transcription factor produced in a hypoxic environment), BNip3 (a HIF-1α regulated pro-apoptotic protein) and TUNEL (detecting DNA fragmentation in apoptosis).

The HIF-1α expression was greatest in mild impingement and in partial, small, medium and large tears. BNip3 expression increased significantly in partial, small, medium and large tears but was reduced in massive tears. Apoptosis was increased in small, medium, large and massive tears but not in partial tears.

These findings reveal evidence of hypoxic damage throughout the spectrum of pathology of the rotator cuff which may contribute to loss of cells by apoptosis. This provides a novel insight into the causes of degeneration of the rotator cuff and highlights possible options for treatment.