Synovial mast cells from knee and hip osteoarthritis: histological study and clinical correlations.

PURPOSE: The aim of this study was to investigate the presence of synovial mast cells (MCs) in hip and knee tissue from osteoarthritis (OA) patients and to correlate them with clinical and radiological data. METHODS: Synovial tissue was obtained during arthroplasty from 60 patients, 30 with knee OA and 30 with hip OA. Control synovial tissue was obtained from 30 patients without OA, 15 undergoing above-knee amputation and 15 receiving a hip replacement for fracture. Before surgery, the radiographic findings were graded according to the Kellgren-Lawrence system and clinical data including pain (VAS) and functional information (KOOS and HOOS) was collected. The tissue was stained with hematoxylin-eosin and toluidine blue for histochemistry and incubated with CD117 and CD31 antibodies for immunohistochemistry. MC and vessel number and synovitis score were determined in all samples. RESULTS: Mean MC number, synovitis score and vessel number were significantly higher in the OA samples (p < 0.05) than in control tissue. MC number correlated with the synovitis score and disease severity in both patient groups. CONCLUSIONS: The prevalence of MCs in synovium from OA patients and their association with synovial inflammation and pain suggest a role for them in OA pathophysiology.

Effects of NGF and BDNF on chondrocytes: a microarray analysis.

Osteoarthritis (OA) represents an inflammation-driven injury of articular tissues, progressively leading to structural and functional joint impairment. The main symptom of OA is pain. Although it has been well established that OA represents a whole joint disease, the source of pain remains to be clarified. Nowadays, it has been well established that neurotrophines expression is evident in joints affected by OA. In addition, elevated NGF levels are found in the synovial fluid of patients with inflammatory or degenerative rheumatic diseases, including OA, rheumatoid arthritis and spondylarthritis. Growing evidences indicate that blocking NGF signaling using an anti NGF agent (i.e. tanezumab) provides effective pain relief. This study analyzed the effects of NGF and BDNF on cultured human chondrocytes by evaluating and their effects on chondrogenesis, chondrocyte differentiation and cartilage degeneration through a microarray analysis. The whole transcriptome analysis performed in this study highlighted how NGF and BDNF could be able to induce a proinflammatory response in human chondrocytes. Moreover, NGF and BDNF treatments seems to be able to induce the activation of several genes involved in the OA pathogenesis as IL17AR, HLA-DRB1, GDF-15, NR1D1, MCF2L and TGF-Beta.

Skeletal muscle repair in a rat muscle injury model: the role of growth hormone (GH) injection.

OBJECTIVE: Muscle injury tends to heal with incomplete functional recovery. Among the growth factors released in the physio-pathological response of muscle lesion, the Insulin-like Growth-Factor-1 (IGF-1) results in an engine factor of the reparation program. The therapeutic use of growth factors has been exploited to improve healing. As IGF-1 is a primary mediator of the effects of growth hormone (GH), we exploited its systemic administration to muscle recovery in a rat model of muscle injury. MATERIALS AND METHODS: Monolateral lesion of the longissimus dorsi muscle of rats was performed. Animals were divided into 5 groups: four groups for histological studies and serum hormone dosage, whilst the fifth group represented the uninjured control. Rat GH was intraperitoneally administered after 24h from the surgical lesion at three different concentrations (0.1, 0.2, 0.4 mg/kg). At 3 days from surgery, immunohistochemical and histological analyses evaluated the expression of MyoD and Myogenin, and the presence of neovascularization and inflammation, respectively. After 2 months, we analyzed the presence of muscle regeneration and fibrosis. RESULTS: The treatment with GH resulted in a significant increase in neovascularization and Myogenin expression at 24h from injury in comparison with the control. This suggested speed up biological recovery times. After two-months, a dose-dependent increase of the connective component was observed. CONCLUSIONS: The potential effect of GH on muscle repair and regeneration, through the activation of satellite cells already demonstrated in vitro, was confirmed in this in vivo experimental approach. This study sheds light on the role of growth factors in damage repair mechanisms to find an appropriate biological treatment for muscle injury.

Synovial and serum levels of NGF in osteoarthritis and rheumatic diseases: a systematic review.

NGF has raised interest as a target molecule in the treatment of OA, after the clinical evidences that antagonization of NGF axis provides symptoms relief in OA. Thus, we conducted a systematic review of the literature to investigate the evidence of NGF being overexpressed during OA. We conducted a database search on Medline using keywords including NGF, serum, synovial fluid, AND osteoarthritis or arthritis. We included study conducted on human, with serum or synovial specimens and an OA cohort. Nine studies met the inclusion criteria. Serum levels ranged from non-detectable to 153.5±28.6 pg/ml. Synovial fluid levels ranged from non-detectable to nearly 210±82 pg/ml. One study supported the evidence of an increased level of NGF in SF and serum of OA patients. The concentration of NGF reported in these studies is controversial and evidence of overexpression of NGF is low.

The metabolic effects of quinolones and steroids on human ligament cells: an in vitro study.

The toxic effects of fluoroquinolones and steroid on tendon cells have been well established, but their role on human ligamentocytes remain unclear. We have investigated the effects of ciprofloxacin and methylprednisolone on human anterior cruciate ligamentocytes after 7 and 14 days of culture. We evaluated cell viability, Annexin V-FITC/PI assay, senescence-associated ß-galactosidase staining, and collagen type I detection. Regarding quinolones administration, we observed that ligament cells treated with ciprofloxacin have characterized by a significantly decrease of cell viability and collagen type I expression and an increase of apoptotic cells. In cells treated with high dose of steroid we observed a significantly decrease of cell viability and collagen type I expression and the presence of senescent cells. Therefore, ciprofloxacin and methylprednisolone might have cytotoxic effects on ligamentocytes by two distinct mechanisms. Quinolones seem to induce cell apoptosis, while steroids might be able to induce cellular senescence. Hence their use should be avoided in athletes and in orthopedic surgery.

Characterization of human costal cartilage: is it an adapt tissue as graft for articular cartilage repair?

Several techniques and different biological or artificial tissues have been proposed as graft to restore articular defects. However, among the numerous and heterogeneous procedures proposed over time, the current literature findings are not conclusive. The aim of the current study is to evaluate if human costal cartilage can be suitable as graft for restoring articular cartilage defects. Knee articular cartilage and costal cartilage samples were obtained respectively from patients that underwent anterior cruciate ligament reconstruction (samples from notch plasty) or knee joint replacement and ear reconstruction or rhinoplasty through rib graft. The samples were stained with hematoxylin eosin, safranine-O, Gomori paraldehyde-fuchsin and Von Kossa for light microscopy. Immunohistochemistry was performed using anti-collagen I, II, IV and anti-SOX9 antibodies. Furthermore, samples were analyzed by transmission electron microcopy (TEM). In both cartilage, the cells are arranged in quite similar layers and the matrix show the same hyaline appearance: presence of type II collagen and solphated glycosaminoglycans, and absence of type I collagen and SOX-9. The bigger difference between the two hyaline tissues is the presence of perichondrium that surrounds all the specimens of costal cartilage. It consists of two separate layers where the inner one seems to get thinner with aging. The results show that rib cartilage seems to be an adapt tissue as graft for articular cartilage repair from a histological point of view. However, to date its therapeutic potential remains to be clearly defined by animal and clinical studies.

Nerve growth factor administration on cultured human ligamentocytes: an in vitro pilot study.

Nerve growth factor (NGF) is involved in several joint pathologies. It has been demonstrated that its concentration increases in synovial fluid and tissue from arthritis. However, its role in joint homeostasis and pathophysiology still remain to be clarified. This study analyzed the effect of 200 ng/ml on cultured human ligamentocytes by evaluating cell proliferation, cell phenotype and gene expression. The MTT test excluded an influence on cell viability at 7 and 14 days. Regarding cell phenotype, we observed that NGF might promote the synthesis of COL1A1. On the other hand, real time PCR showed that NGF did not influence gene expression of COL3A1, FGF-BETA, IGF1, MMP2, MMP3, MMP9 and MMP13. However, COL1A1 gene was significantly upregulated in treated cell at 14 days. Our results suggest that NGF may have an anabolic effect on ligament. Additional investigations are necessary to determine how NGF may influence ligamentocytes..

Effect of nerve growth factor on cultured human chondrocytes.

Nerve growth factor (NGF) is involved in several joint diseases. It participates in nociception and neurogenic inflammation and its concentrations increase in synovial fluid and tissue from arthritis. However, data about its role in articular cartilage are scant and conflicting. This study analysed effects of different NGF concentrations on cultured human chondrocytes by evaluating cell proliferation, cell phenotype, and gene expression. The MTT test excluded an influence on cell viability. Alcian blue and S100 staining demonstrated that NGF may induce de-differentiation of the chondrocyte phenotype. Real-time PCR showed that NGF did not influence gene expression of type I, II and XI collagen, TGF-ß, IGF-1 and metalloproteinase (MMP)-13, while it reduced the expression of MMP-3. These findings show that NGF may have uncertain effects in human chondrocytes. Further investigations by wider gene expression and protein synthesis analyses are required to determine how chondrocytes may be influenced by NGF.

EFFECT OF PLATELET RICH PLASMA CONCENTRATION ON SKELETAL MUSCLE REGENERATION: AN EXPERIMENTAL STUDY.

Skeletal muscle injuries are common causes of severe long-term pain and physical disability, accounting for up to 55% of all sports injuries. The phases of the healing processes after direct or indirect muscle injury are complex but clearly defined and include well-coordinated steps: degeneration, inflammation, regeneration, and fibrosis. Despite this frequent occurrence and the presence of a body of data on the pathophysiology of muscle injuries, none of the current treatment strategies have shown to be really effective in strictly controlled trials. Platelet-rich plasma (PRP) is a promising alternative approach based on the ability of autologous growth factors (GFs) to accelerate tissue healing, improve muscular regeneration, increase neovascularization and reduce fibrosis. The present study is focused on the use of different concentrations of PRP as a source of GFs. Unilateral muscle lesions were created on the longissimus dorsi muscle of Wistar rats. Twenty-four h after surgical trauma, the lesion was filled with an intramuscular injection of PRP at 2 different concentrations. A group of rats were left untreated (controls). Animals were sacrificed at 3, 15 and 60 days from surgery. Histological, immunohistochemical and histomorphometric analyses were performed to evaluate muscle regeneration, neovascularization, fibrosis and inflammation. The PRP-treated muscles showed better muscle regeneration, more neovascularization and a slight reduction of fibrosis compared with the control muscles in a dose dependent manner. However, further studies also assessing pain and functional recovery are scheduled.

One-step cartilage repair in the knee: collagen-covered microfracture and autologous bone marrow concentrate. A pilot study.

BACKGROUND: Different single-stage surgical approaches are currently under evaluation to repair cartilage focal lesions. To date, only little is known on even short-term clinical follow-up and almost no knowledge exists on histological results of such treatments. The present paper aims to analyze the clinical and histological results of the collagen-covered microfracture and bone marrow concentrate (C-CMBMC) technique in the treatment of focal condylar lesions of knee articular cartilage. METHODS: Nine patients with focal lesions of the condylar articular cartilage were consecutively treated with arthroscopic microfractures (MFX) covered with a collagen membrane immersed in autologous bone marrow concentrate (BMC) from the iliac crest. Patients were retrospectively assessed using several standardized outcome assessment tools and MRI scans. Four patients consented to undergo second look arthroscopy and biopsy harvest. RESULTS: Every patient was arthroscopically treated for a focal condylar lesion (mean area 2.5 SD(0.4) cm(2)). All the patients (mean age 43 SD(9) years) but one experienced a significant clinical improvement from the pre-operative condition to the latest follow-up (mean 29 SD(11) months). Cartilage macroscopic assessment at 12 months revealed that all the repairs appeared almost normal. Histological analysis showed a hyaline-like cartilage repair in one lesion, a fibrocartilaginous repair in two lesions and a mixture of both in one lesion. CONCLUSIONS: The first clinical experience with single-stage C-CMBMC for focal cartilage defects in the knee suggests that it is safe, it improves the short-term knee function and that it has the potential to recreate hyaline-like cartilage.

Vitamin MK-7 enhances vitamin D3-induced osteogenesis in hMSCs: modulation of key effectors in mineralization and vascularization.

The osteoblast is the bone-forming cell and is derived from mesenchymal stem cells (MSCs). Osteo-inductive substances could represent a useful therapeutic approach during the fracture repair process. The aim of this work was to evaluate the effects of vitamin MK-7, alone or in association with vitamin D3, in differentiating human MSCs (hMSCs) in vitro along the osteoblastic lineage. In particular, primary endpoints of the study include gene and protein markers of osteoblast differentiation. Considering genes involved in bone formation and mineralization, our data show that vitamin MK-7 enhances vitamin D3 gene induction of osteocalcin (OC). Among genes related to cell growth and differentiation, a specific effect of vitamin MK-7 was observed for growth differentiation factor-10 (GDF10) and insulin-like growth factor 1 (IGF1), the latter being also involved in the induction of vascular endothelial growth factors (VEGFA). Accordingly, vitamin co-supplementation greatly affected VEGFA and its receptor fms-related tyrosine kinase 1 (FLT1), a key factor in both angiogenic and osteogenic processes. These results stress the relevance of MK-7 and D3 co-supplementation in the bone-healing process as able to modulate the expression of genes involved in both mineralization and angiogenesis. Moreover, at the protein level co-association of vitamins might provide an optimal balance between induction and carboxylation of osteocalcin, essential for its functionality in the extracellular matrix (ECM). Our results may provide hints for therapeutic application of hMSCs in bone disease, clarifying mechanisms involved in stem cell-mediated bone development, and they also highlight the relevance of co-supplementation strategies, since single supplementations might result in a suboptimal effect.

Mixed type I and type II collagen scaffold for cartilage repair: ultrastructural study of synovial membrane response and healing potential versus microfractures (a pilot study).

The association between microfracture of the subchondral plate and a coverage scaffold has emerged as a promising strategy to treat cartilage lesions in a one-step procedure. Between different types of scaffolds (e.g. collagen, hyaluronic acid, polyglycolic acid) currently studied, type I collagen scaffold is the most used for this purpose, and is currently adopted for humans. The aim of this study was to test a novel scaffold made of mixed type I and II collagen (I-IICS) in order to define the immunological reaction of the synovial tissue and the repair capabilities induced by the collagen membrane when associated with microfracture. Eight New Zealand White rabbits, aged 180 days, were operated on bilaterally on the medial femoral condyle. A circular cartilage lesion was performed up to the calcified layer of the medial femoral condyle, and the centre of the lesion was microfractured. Randomly, one of the two lesions was covered with the I-IICS (treated), and the other was left uncovered (control). The synovial membrane reaction and the quality of the cartilage tissue repair were investigated at 2, 90, 180 and 270 days macroscopically, histomorphologically and ultrastructurally. Expression of tumor necrosis factor-alpha (TNF-alpha) in synovial tissue by immunocytochemistry analyses was also investigated. In the control group, at 2 days gold particles were localized mainly on synoviocyte type A, less on synoviocytes type B and on collagen bundles; in the treated group the reaction is more intense in cells in the matrix, but at 180 days controls and treated joints were very similar. The synovial membranes of the joints receiving the I-IICS did not reveal significant changes compared to the age-matched controls. Signs of inflammation were present at the 90-day time-point, and became less evident at afterwards. The degradation of the scaffolds was already evident at the 90-day time-point. The quality of the cartilage repair of the rabbits treated with the I-IICS was slightly better in 5 cases out of 6 in comparison to the controls. However, a statistically significant difference was not detected (p=0.06). Scaffolds made of mixed type I and II collagen exhibited good biocompatibility properties in vivo and favoured cartilage restoration when associated with microfracture, as shown in this pilot study.

Single-stage cartilage repair in the knee with microfracture covered with a resorbable polymer-based matrix and autologous bone marrow concentrate.

BACKGROUND: Different single-stage surgical approaches are currently under evaluation to repair focal cartilage lesions. This study aims to analyze the clinical and histological results after treatment of focal condylar articular lesions of the knee with microfracture and subsequent covering with a resorbable polyglycolic acid/hyaluronan (PGA -HA) matrix augmented with autologous bone marrow concentrate (BMC). METHODS: Nine patients with focal lesions of the condylar articular cartilage were consecutively treated with arthroscopic PGA -HA-covered microfracture and bone marrow concentrate (PGA -HA-CMBMC). Patients were retrospectively assessed using standardized assessment tools and magnetic resonance imaging (MRI). Five patients consented to undergo second look arthroscopy and 2 consented biopsy harvest. RESULTS: All the patients but one showed improvement in clinical scoring from the pre-operative situation to the latest follow-up (average 22±2months). The mean IKDC subjective score, Lysholm score, VAS and the median Tegner score significantly increased from baseline to the latest follow-up. Cartilage macroscopic assessment at 12months revealed that one repair appeared normal, three almost normal and one appeared abnormal. Histological analysis proofed hyaline-like cartilage repair tissue formation in one case. MRI at 8 to 12months follow-up showed complete defect filling. CONCLUSIONS: The first clinical experience with single-stage treatment of focal cartilage defects of the knee with microfracture and covering with the PGA -HA matrix augmented with autologous BMC (PGA -HA-CMBMC) suggests that it is safe, it improves knee function and has the potential to regenerate hyaline-like cartilage. LEVEL OF EVIDENCE: IV, case series.

Platelet rich fibrin matrix effects on skeletal muscle lesions: an experimental study.

Even though muscle injuries are very common, few scientific data on their effective treatment exist. Growth Factors (GFs) may have a role in accelerating muscle repair processes and a currently available strategy for their delivery into the lesion site is the use of autologous platelet-rich plasma (PRP). The present study is focused on the use of Platelet Rich Fibrin Matrix (PRFM), as a source of GFs. Bilateral muscular lesions were created on the longissimus dorsi muscle of Wistar rats. One side of the lesion was filled with a PRFM while the contralateral was left untreated (controls). Animals were sacrificed at 5, 10, 40 and 60 days from surgery. Histological, immunohistochemical and histomorphometric analyses were performed to evaluate muscle regeneration, neovascularization, fibrosis and inflammation. The presence of metaplasia zones, calcifications and heterotopic ossification were also assessed. PRFM treated muscles exhibited an improved muscular regeneration, an increase in neovascularization, and a slight reduction of fibrosis compared with controls. No differences were detected for inflammation. Metaplasia, ossification and heterotopic calcification were not detected. This preliminary morphological experimental study shows that PRFM use can improve muscle regeneration and long-term vascularization. Since autologous blood products are safe, PRFM may be a useful and handy product in clinical treatment of muscle injuries.

Use of collagen scaffold and autologous bone marrow concentrate as a one-step cartilage repair in the knee: histological results of second-look biopsies at 1 year follow-up.

Chondral articular defects are a key concern in orthopaedic surgery. To overcome the disadvantages of autologous chondrocyte implantation (ACI) and to improve the outcomes of autologous matrix-induced chondrogenesis (AMIC), the latter technique is currently augmented with bone marrow concentrate injected under or seeded onto the scaffold. However, to date, only a little is known about histological outcomes of either the AMIC technique or AMIC associated with bone marrow concentrate. This study aimed to evaluate the quality of the repair tissue obtained from biopsies harvested during second-look arthroscopy after arthroscopic AMIC augmented with bone marrow concentrate. We analysed five second-look core biopsies harvested at 12 months follow-up. At the time of biopsy the surgeon reported the quality of the repair tissue using the standard ICRS Cartilage Repair Assessment (CRA). Every biopsy together with patient data was sent to our centre to undergo blind histological evaluation (ICRS II Visual Histological Assessment Scale) and data analysis. Five asymptomatic patients (mean age 43.4 years) had isolated lesions (mean size was 3.7 cm2) at the medial femoral condyle. All the implants appeared nearly normal (ICRS CRA) at arthroscopic evaluation and had a mean overall histological (ICRS II) of 59.8±14,5. Hyaline-like matrix was found in only one case, a mixture of hyaline/fibrocartilage was found in one case and fibrocartilage was found three cases. Our clinical and histological data suggest that this procedure achieved a nearly normal arthroscopic appearance and a satisfactory repair tissue, which was possibly still maturing at 12 months follow-up. Further studies are needed to understand the true potential of one-step procedures in the repair of focal chondral lesions in the knee.

Blood exposure has a negative effect on engineered cartilage.

PURPOSE: The aim of this study was to investigate the in vitro effect of different concentrations of blood on the morphological and biochemical properties of engineered cartilage. Previous studies have demonstrated a negative effect of blood on native cartilage; however, the effect of the contact of blood on engineered cartilage is unclear. METHODS: Articular chondrocytes were isolated from swine joints, expanded in monolayer culture, and seeded onto collagen membranes. The seeded membranes were cultured for 3 days in the presence of different concentrations of peripheral blood. Some samples were retrieved at the end of the blood contact, others after 21 additional days of standard culture conditions, in order to investigate the "long-term effect" of the blood contact. RESULTS: All seeded samples showed an increase in the weight and an evident cartilage-like matrix production. A concentration-dependent reduction in the mitochondrial activity due to blood contact was shown at the earlier culture time, followed by a partial recover at the longer culture time. CONCLUSION: A blood contact of 3 days affected the chondrocytes' activity and determined a delay in the maturation of the engineered cartilage. These findings have clinical relevance, as autologous chondrocytes seeded onto biological scaffolds has become an established surgical method for articular cartilage repair. Therefore, further investigation into material sciences should be encouraged for the development of scaffold protecting the reparative cells from the blood insult.

Osteoinduction properties of different growth factors on cells from non-union patients: in vitro study for clinical application.

This report compares the effect of rhBMPs and PRG on cells derived from human non-union sites. Treatment of non-union continues to be a challenging task for the trauma surgeon often resulting in unsatisfactory results and long-term morbidity. Over the past two decades, the possibility to use growth factors in bone regeneration has been investigated. In this study we compared the in vitro capability of two recombinant human bone morphogenetic proteins (rhBMP-2 and rhBMP-7) and activated platelet-rich plasma (PRG) to stimulate proliferation and/or differentiation of cells derived from non-union patients. Cells derived from the lesion sites, osteoblasts and mesenchymal stem cells (MSCs) derived from other bone sites of the same patients were used. Treatment with rhBMP-7 or rhBMP-2 showed an improvement in the expression of osteoblastic markers (osteonectin and osteocalcin) in cells derived from human non-union sites. This enhancement was more marked in MSCs, while no significant changes were observed in osteoblast cultures. The PRG treatment produced in all analysed samples a considerable increase in cell proliferation without affecting cell differentiation. On the basis of our results, for an effective biological treatment of non-unions, small amounts of autologous bone marrow (MSCs) are necessary in the lesion site in order to provide both growth factors and a sufficient number of responsive cells. Finally, our results prove that sequential timing administration of PRG and rhBMPs may be used in new therapeutic strategy.

Dexamethasone affects Fas- and serum deprivation-induced cell death of human osteoblastic cells through survivin regulation.

Glucocorticoid-induced bone loss is the most prevalent form of secondary osteoporosis. Such loss could be due to the alteration of osteoclast and osteoblast lifespan through regulated apoptosis. The current study investigated the effect of dexamethasone on Fas- and starvation-induced apoptosis of mature osteoblasts and their precursors. Using the human osteoblastic hFOB1.19 and the MG63 osteosarcoma cell lines, we found that sub-lethal doses of dexamethasone act on pre-osteoblasts but not on mature cells by increasing their susceptibility to apoptosis. Apoptosis occurs in a caspase-dependent manner as both DNA fragmentation and mitochondrial transmembrane potential dissipation (ΔΨm) are inhibited by the pan-caspase inhibitor zVAD. The increased susceptibility of osteoblast precursors to apoptosis could be due to dexamethasonemediated down-regulation of survivin expression. Dexamethasone can up-regulate survivin, and to a lesser extent Bcl-2, in mature cells but not in pre-osteoblasts. In addition, it can induce FLIP over-expression in osteosarcoma cells. All these effects are inhibited by the glucocorticoid antagonist RU486, indicating that dexamethasone action is specific and, furthermore, that it depends on glucocorticoid receptor. Finally, we have found that survivin and Bcl-2 are essential for pre- and mature osteoblast survival as their silencing is sufficient to induce spontaneous apoptosis in both cell types. In conclusion, our data outline a new molecular mechanism of glucocorticoid-mediated bone loss due to the enhanced apoptosis of precursors compared to mature osteoblasts. Furthermore, the data suggest a mechanism of dexamethasone-induced resistance of osteosarcoma cells to Fas- and stress-induced apoptosis.

Adult mesenchymal stem cells for bone and cartilage engineering: effect of scaffold materials.

Bone marrow is a useful cell source for skeletal tissue engineering approaches. In vitro differentiation of marrow mesenchymal stem cells (MSCs) to chondrocytes or osteoblasts can be induced by the addition of specific growth factors to the medium. The present study evaluated the behaviour of human MSCs cultured on various scaffolds to determine whether their differentiation can be induced by cell-matrix interactions. MSCs from bone marrow collected from the acetabulum during hip arthroplasty procedures were isolated by cell sorting, expanded and characterised by a flow cytometry system. Cells were grown on three different scaffolds (type I collagen, type I + II collagen and type I collagen + hydroxyapatite membranes) and analysed by histochemistry, immunohistochemistry and spectrophotometry (cell proliferation, alkaline phosphatase activity) at 15 and 30 days. Widely variable cell adhesion and proliferation was observed on the three scaffolds. MSCs grown on type I+II collagen differentiated to cells expressing chondrocyte markers, while those grown on type I collagen + hydroxyapatite differentiated into osteoblast-like cells. The study highlighted that human MSCs grown on different scaffold matrices may display different behaviours in terms of cell proliferation and phenotype expression without growth factor supplementation.

Vitamin K and D association stimulates in vitro osteoblast differentiation of fracture site derived human mesenchymal stem cells.

There is growing interest in osteoinductive agents for fracture healing especially in patients with non-union or delayed-union fractures. The aim of the present study is the assessment of the association of Vitamins D3 and K1 on proliferation and differentiation of human mesenchymal stem cells (hMSCs) derived from fracture sites in view of a possible clinical use. The synergic effect of Vitamin D3 and Vitamin K2 in preventing osteoporosis has been documented in clinical practice; however no reports investigating this association for fracture healing are present. Our data show a different outcome on cell proliferation linked to the different timing of drug administration as well as a synergic effect of the two vitamins on cell differentiation. The high level of osteocalcin and carboxylated osteocalcin detected in hMSCs treated with the association of the two vitamins in comparison with controls and with single vitamin administration underline the differentiation of these cells into osteoblastic phenotype. Our results indicate for the first time that vitamin D3 and K1 association is able to modulate in vitro the differentiation towards osteoblastic phenotype of hMSCs derived from fracture sites, thus offering clinicians a promising and low-cost strategy for reparative osteogenesis.