Nerve growth factor (NGF) is involved in several joint diseases. It participates in pain initiation, inadequate nociception and neurogenic inflammation; its concentrations are increased in synovial fluid and tissue from human and experimental arthritis. However, data about its role in normal and pathological articular cartilage are scant and conflicting. This study assesses the effects of different

NGF concentrations on cultured healthy human chondrocytes by evaluating cell proliferation, cell phenotype, and gene expression.

The 3-[4,5-dimethylthiazol-2-y1]-2,5-diphenyl-2H-tetrazolium bromide (MTT) test excluded an influence on cell viability; alcian blue and S100 staining demonstrated that NGF induced de-differentiation of the chondrocyte phenotype; real-time PCR disclosed that it reduced the expression of collagen type II (COL2A1) and transforming growth factor-β (TGF-β), key factors involved in articular cartilage integrity, and stimulated upregulation of metalloproteinase (MMP)-3 and MMP-13.

These findings suggest that NGF may adversely affect differentiated chondrocytes from articular cartilage by inhibiting the expression of the collagens found in normal articular cartilage (COL2A1), while exerting a degradative effect though TGF-β downregulation and MMP-13 and MMP-3 upregulation. Further investigation is required to determine whether the gene expression pattern found in our study is associated with changes in protein expression.

Summary Statement

This experimental study showed that platelet rich fibrin matrix can improve muscle regeneration and long-term vascularization without local adverse effects.

Aims: The use of vitamin K was proposed in the treatment of osteoporosis. Some experimental studies suggested that vitamin K might promote mesenchymal stem cells (MSCs) differentiation into osteoblasts progenitors and inhibit osteoclasts formation. In the present study we analysed the effects of vitamin K at different concentrations on human mesenchymal stem cells derived from fracture callus.

Methods: MSCs were obtained from the fracture’s site of three patients during surgical operation of osteosynthesis. Cells were grown on plastic plates in DMEM, 10% foetal bovine serum (FBS), 1% penicillin-streptomycin, 1% fungizone, 5mM beta-glycerophosphate and 50 microg/ml ascorbic acid. Half of the samples was incubated with vitamin D (10 nm) and K at different concentrations (1, 3, 10 microM). Proliferation rate (MTT colorimetric assay) and cell differentiation (FACSCalibur flow cytometry) were assessed at 3, 10 and 20 days. Immunocytochemical analysis (not-carboxylated osteocalcin and carboxylated osteocalcin) was also performed.

Results: MSCs stimulated with vitamin K and D expressed higher levels of osteoblastic markers than controls at 3, 10 and 20 days of colture.

Conclusions: This study confirmed the results obtained in previous in vitro experiments: vitamin K has osteoinductive properties on MSCs derived from fracture callus and it could play a role in reparative osteogenesis in vivo.

Aims: A new type I collagen membrane developed for use as a tendon graft was tested in vitro and in vivo.

Methods: The membrane (Opocrin, Italy) is obtained from type-I collagen harvested from equine Achilles tendon and is composed of collagen I fibres oriented in a single direction. It is isotropic, resorbable, hygroscopic and non immunogenic acellular membrane.

Primary human fibroblasts were seeded on collagen I membranes with aligned fibres (# 40133) with and randomly arranged fibres (# 40153). Cell proliferation was evaluated at 4, 8 and 12 days by spectrophotometry. Membrane sections were studied by immunohistochemistry and by confocal microscope on day 12 of culture.

The middle third of the patellar tendon was lesioned bilaterally in 10 adult male New Zealand White rabbits and repaired on the right side by a graft (# 40133). The contralateral tendon was left untreated and served as control. Animals were euthanized 1 or 6 months after surgery and the tendon grafts subjected to histological examination.

Results: The in vitro study demonstrated cell viability and proliferation already on day 4 from membrane seeding.

Cells were homogeneously distributed, with a more marked orientation along the main membrane axis in batch 40133 than in 40153. The in vivo study showed that cell orientation and differentiation in the scaffold with aligned fibres was satisfactory, with decreased cellularity, good integration with the surrounding tissue and crimp formation. Inflammatory reaction or excessive implant neovascularization were never observed.

Conclusion: The new type I collagen membrane exhibited a behaviour similar to other tendon or ligament scaffolds. Fibre orientation in the membrane with aligned fibres allowed to obtain a quick and well-oriented cell growth. The membrane appears to be suitable for application in tendon and ligament repair and substitution.

The search for bone substitutes has stimulated the study of growth factors with osteoinductive properties. Bone morphogenetic proteins (BMPs) have been shown to have a central role in endochondral and intramembranous bone formation and are thought to promote normal bone healing. Recent studies demonstrated that platelet-rich plasma (PRP) can provide several growth factors and stimulate osteogenesis. The aim of the present study was to analyse the in vitro effects of rhBMP-7 and PRP on phenotype and proliferation of cells from the site of non-union and from non-affected bone.

During the surgical treatment of seven cases of non-union, normal cancellous bone and tissue from the non-union site were harvested. Osteoblast-like cells and fibroblast-like cells were isolated and characterised. Mesenchymal cells were obtained from bone marrow of the same patients. Each cell type was incubated with rhBMP-7 and PRP at different concentrations. Proliferation rate and alkaline phosphatase activity were assessed at 3, 7, 15 and 30 days. Histochemical and immunohistochemical analyses were performed at 15 and 30 days.

The proliferation rate of osteoblast-like cells and mesenchymal cells wasalways higher than that of fibroblast-like cells from the non-union site. Growth factors induced mesenchymal cells to express osteoblast phenotype markers.

The results suggest that fibroblast-like cells from the site of non-union are poorly responsive to growth factors, even at highest stimulation. In surgical practice these data strongly suggest adding osteoblast-like cells and mesenchymal cells from non-affected sites at the non-union site to enhance the osteogenic response to growth factors.

Bone marrow would represent a useful source of cells for skeletal tissue engineering. Marrow mesenchymal stem cells (MSC) can generate cartilage, bone and fat. The differentiation of this multipotent population into fibroblast, chondrocytes or osteoblasts can be inducted in vitro by the addiction of growth factor like bFGF, TGFA7, BMP-2.

In order to evaluate the possibility of inducing cell differentiation by cell-matrix interaction, we studied the in vitro behaviour of human MSC cultured on various scaffolds.

Bone marrow was obtained during surgery for pelvic fractures or hip arthroplasty. MSC were isolated by cell sorting (CD45/glycophorin A micromagnetic beads), expanded and characterised by FACSCalibur flow cytometry system (CD3, CD34, CD14, CD45, CD90 and CD105). Then cells were grown for 30 days on different scaffolds: type I and type II collagen, type I collagen + hydroxyapatite. Histochemical (alcian blue, safranin O, ALP and von Kossa stains), immunohistochemical (type I e II collagen, chondroitin sulphate, osteonectin), histomorphometric (area %) and spectrophotometric (cell proliferation, PG synthesis, ALP activity) analyses were performed after 15 and 30 days of culture.

Among the scaffolds tested in the present study, we observed a great variability in terms of MSC adhesion and proliferation. MSC grown on type II collagen differentiated into cells expressing chondrocytes markers (S100, collagen II, chondroitin-S). MSC grown on type I collagen + hydroxyapatite differentiated into osteoblast-like cells.

These data evidenced that MSC-matrix interaction can influence phenotype expression, cell adhesion and growth rate.