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

Aims. Autologous bone graft (ABG) is considered the ‘gold standard’ among graft materials for bone regeneration. However, complications including limited availability, donor site morbidity, and deterioration of regenerative capacity over time have been reported. P-15 is a synthetic peptide that mimics the cell binding domain of Type-I collagen. This peptide stimulates new bone formation by enhancing osteogenic cell attachment, proliferation, and differentiation. The objective of this study was to conduct a systematic literature review to determine the clinical efficacy and safety of P-15 peptide in bone regeneration throughout the skeletal system. Methods. PubMed, Embase, Web of Science, and Cochrane Library were searched for relevant articles on 13 May 2023. The systematic review was reported according to the PRISMA guidelines. Two reviewers independently screened and assessed the identified articles. Quality assessment was conducted using the methodological index for non-randomized studies and the risk of bias assessment tool for randomized controlled trials. Results. After screening, 28 articles were included and grouped by surgical indication, e.g. maxillofacial procedures (n = 18), spine (n = 9), and trauma (n = 1). Published results showed that P-15 peptide was effective in spinal fusion (n = 7) and maxillofacial (n = 11), with very few clinically relevant adverse events related to P-15 peptide. Conclusion. This systematic literature review concluded that moderate- (risk of bias, some concern: 50%) to high-quality (risk of bias, low: 46%) clinical evidence exists showing equivalent safety and efficacy in bone regeneration using a P-15 peptide enhanced bone graft substitute compared to ABG. P-15 peptide is safe and effective, resulting in rapid bone formation with a low probability of minor complications. Cite this article: Bone Joint Res 2025;14(2):77–92

Measurements of biochemical markers of bone turnover have been explored as a diagnostic tool for the detection of osteolysis after THA, but their predictive value in individual subjects has been poor. One explanation for this low diagnostic utility is that the mechanism of bone resorption in osteolysis may be different to that occurring in other high bone turnover states, such as osteoporosis, where these markers were principally developed. The aim of this study was to examine the role of the biomarkers urinary ααCTX-I and serum CTX-MMP, that are released in pathological rather than physiological bone turnover states, for detecting periprosthetic osteolysis in a case control study of 23 subjects with osteolysis and 26 controls. All samples were collected between the hours of 0800 and 1000 following an overnight fast, and were assayed using standard techniques. The demographic characteristics of the subjects in both groups were similar. Serum CTX-MMP was greater in the osteolysis versus the control group (P=0.001). Urinary ααCTX-I was similar between osteolysis and control groups (P> 0.05). A cut-off value of 5.50ng/mL CTX-MMP had a sensitivity of 91% (95% CI: 72 to 99) and specificity of 69% (48 to 96) detecting osteolysis (P=0.001). The same cut-off had a sensitivity of 100% (100 to 100) and specificity of 63% (44 to 79) for detecting femoral osteolysis (P=0.0004), and a sensitivity of 89% (65 to 98) and specificity of 58% (39 to 75) for identifying pelvic osteolysis (P=0.014). Serum CTX-MMP shows promise for further investigation as a sensitive bio-marker for detecting periprosthetic osteolysis.

Abstract. Objectives. Human articular cartilage chondrocytes undergo changes to their morphology and clustering with cartilage degeneration as occurs in osteoarthritis. (1). The consequences of chondrocyte de-differentiation on mechanically-resilient extracellular matrix metabolism are, however, unclear. We have assessed whether there is a relationship between abnormal chondrocyte morphology, as demonstrated by the presence of cytoplasmic processes, and chondrocyte clustering, with cell-associated type-I collagen during cartilage degeneration. Methods. The femoral heads of 9 patients were obtained (with Ethical permission/consent) following hip replacement surgery and cartilage areas graded (Grade-0 non-degenerate; Grade-1 mildly degenerate). In situ chondrocyte morphology and cell-associated type-I collagen were labelled fluorescently with CMFDA Cell tracker green, and immuno-fluorescence respectively then visualised/quantified using confocal laser scanning microscopy and imaging software. Results. When comparing data from 9 femoral heads with Grade-0 [N(n)=6(72)] or Grade-1 cartilage [(N(n)=9(108)], the latter had a higher percentage of chondrocytes with cytoplasmic processes (length >5µm) (P=0.018) and clusters (≥5 cells within a lacuna) (P<0.001). The percentage of chondrocytes with processes and clusters displaying cell-associated type-I collagen, was also higher in degenerate cartilage (P<0.001 for both). However, some morphologically-normal chondrocytes exhibited cell-associated collagen type-I labelling while some clusters did not label with collagen type-I. Intriguingly, even in Grade-0 cartilage, some chondrocytes were morphologically abnormal and exhibited cell-associated type-I collagen. Conclusions. These results suggest a complex relationship between chondrocyte morphology/clusters and cell-associated collagen type-I. The presence of this collagen type implies chondrocyte de-differentiation to a fibroblastic phenotype even in non-degenerate cartilage. This cell type produces a fibro-cartilaginous ‘repair’ matrix which is considerably weaker than the collagen type-II matrix of healthy hyaline cartilage and may give rise to focal points of mechanical weakness. Funder. Chief Scientist's Office, Scotland (Grant TCS/18/01). Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Cartilage and bone degeneration are major healthcare problems affecting millions of individuals worldwide. Elucidation of the processes modulating the cell-matrix interactions involved in cartilage or bone formation offer tremendous potential in the development of clinically relevant strategies for cartilage and bone regeneration. We have therefore adopted an ex vivo tissue engineering approach to investigate chondrogenesis and osteogenesis using a mix human mesenchymal progenitor populations encapsulated in biomineralised polysac-charide templates with or without the addition of type-I collagen. Alginate/chitosan polysaccharide capsules containing 2.5mg/ml type-I collagen and TGF-beta-3 were encapsulated with human bone marrow cells (HBMC), articular chondrocytes or a co-culture at a ratio of 2:1 respectively and placed in a rotating (Synthecon) biore-actor or held in static 2D culture conditions for 28 days, to determine whether the presence of type-I collagen within the alginate could promote the synthesis of an extracellular matrix. Constructs were stained with alcian blue, sirius red and von Kossa. In bioreactor samples encapsulated with HBMC and type-I collagen, viable cells were present within lacunae, surrounded by a matrix of proteo-glycans and fibrous collagen, which was mineralized. Immunohistochemistry and polarised light microscopy indicated an organised collagenous matrix with extensive expression of type I collagen and bone sialoprotein with small regions of type II collagen. Type X collagen was also expressed indicating the presence of hypertrophic chondrocytes. Within the static HBMC groups, smaller areas of matrix were generated with decreased expression of type-I and type-II collagen. Co-culture bioreactor samples also demonstrated regions of new mineralised bone matrix; however these were less prominent than in the HBMC only groups. No matrix formation was observed in chondrocyte cultures although the cells remained viable as assessed by live/dead staining. Biochemical analysis indicated significantly increased (p< 0.05) DNA in all bioreactor samples in comparison with static constructs and significantly increased protein in HBMC bioreactor constructs in comparison with other cell types. These studies outline a unique tissue engineering approach, utilizing individual and mixed human mesen-chymal progenitor populations coupled with innovative polysaccharide templates containing type I collagen and bioreactor systems to promote chondrogenic and osteo-genic differentiation

Abstract. OBJECTIVE. Changes in subchondral bone are one of few disease characteristics to correlate with pain in OA. 1. Profound neuroplasticity and nociceptor sprouting is displayed within osteoarthritic (OA) subchondral bone and is associated with pain and pathology. 2. The cause of these neural changes remains unestablished. Correct innervation patterns are indispensable for bone growth, homeostasis, and repair. Axon guidance signalling factor, Sema3A is essential for the correct innervation patterning of bony tissues. 3. , expressed in osteocytes. 4. and known to be downregulated in bone OA mechanical loading. 5. Bioinformatic analysis has also shown Sema3a as a differentially expressed pathway by bone in human OA patients. 6. HYPOTHESIS: Pathological mechanical load and inflammation of bone causes dysregulation of Sema3A signalling leading to perturbed sensory nerve plasticity and pain. METHODS. Human KOLF2-C1 iPSC derived nociceptors were generated by TALEN-mediated insertion of transcription factors NGN2+Brn3A and modified chambers differentiation protocol to produce nociceptor-like cells. Nociceptor phenotype was confirmed by immunocytochemistry. Human Y201-MSC cells were embedded in 3D type-I collagen gels (0.05 × 106 cell/gel), in 48-well plates and silicone plates, were differentiated to osteocytes for 7 days before stimulation with IL-6 (5ng/ml) and soluble IL-6 receptor (sIL-6r (40ng/ml), IL6/sIL6r and mechanical load mimetic Yoda1 (5μM) or unstimulated (n=5/group) (48-well plates) or were mechanically loaded in silicone plates (5000μstrain, 10Hz, 3000 cycles) or not loaded (n=5/group). Conditioned media transfer was performed from osteocyte to nociceptor cultures assessed by continuous 24-hour phase contrast confocal microscopy. 24-hours after stimulation RNA was quantified by RT-qPCR (IL6) or RNAseq whole transcriptome analysis/DEseq2 analysis (Load). Protein release was quantified by ELISA. Normally distributed data with homogenous variances was analysed by two-tailed t test. RESULTS. IPSC-derived nociceptor-like cells display elongated (>5mm) dendritic projections and nociceptive molecular markers such as TUJ1, PrPH and Neun and TrkA. Sema3A signalling ligands were expressed in 100% of osteocyte cultures. Mechanical loading regulated the Sema3 pathway; Sema3A (0.4-fold, p<0.001), Sema3B (13-fold, p<0.001), Sema3C (0.4-fold, p<0.001). Under inflammatory stimulation by IL6/IL6sR, SEMA3A (7-fold, p=0.01) and receptor Plexin1 (3-fold, p=0.03) show significant regulation. Sema3A protein release showed a significant downregulation of Sema3A release by IL6/sIL6r+Yoda1 (2-fold, p=0.02). Continuous 24-hour phase contrast confocal microscopy measuring the number of extending/retreating dendritic projections revealed that sensory nerve cultures exposed to media from osteocytes stimulated with IL-6/sIL-6R+Yoda1 displayed significantly more invading dendritic projections (p=0.0175, 12-fold±SEM 3.5) across 3 random fields of view within a single stimulated neural culture and significantly fewer retracting dendritic projections (p=0.0075, 2-fold±SEM 0.33) compared to controls. CONCLUSIONS. Here we show osteocytic regulation of Sema3A under pathological mechanical loading and the ability of media pathologically loaded osteocyte cultures to induce the branching and invasion of cultured nociceptor-like cells as displayed in OA subchondral bone. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

The aim of this work was the structural investigation of different type I collagen isoforms at atomic and nanoscale, as well as the evaluation of the impact of different fabrication treatments on the structural, mechanical and biological properties of collagen-based films. Raw type-I collagens from bovine hide (Typ-BH, CS, SYM) and equine tendon (TypE, TypCH and OPO) were analyzed. Materials were then used for fabricating air-dried films, obtained by: 1) dissolution in distilled water (HH); 2) dissolution in acidic medium (AA); 3) homogenization of acid solubilized fibers (HOM). Crosslinking treatments (DHT, DHT+EDC) were also adopted and studied. Analysis by Wide Angle (WAXS) and Small Angle (SAXS) X-ray Scattering was carried out at the XMI L@b (CNR-IC-Bari); Fourier Transform-IR and biological analysis was performed at UniSalento. WAXS and SAXS data on raw materials demonstrated the preferential orientation of collagen molecules and the preservation of hierarchical nanoscale architecture in equine tendon-derived collagens, in particular in chemically extracted, while randomly oriented molecules were found in bovine dermis collagens, together with a certain degree of salt contamination. Concerning equine collagen, we found that TypCH structure is influenced by crosslinking procedures at atomic scale, whereas both processing conditions and crosslinking treatments affect TypE collagen structure at atomic and nanoscale. WAXS, SAXS and FT-IR analyses showed that the HOM processing was the one which ensures a high content of structural super-organization of collagen into triple helices and a high crystalline domainof the final material. Crosslinking of the films by DHT/EDC combined treatment was shown to affect their mechanical stiffness, the latter depending on the collagen source and the specific processing conditions

Tendon injuries are common and current therapies often are unsuccessful. Cell-based therapy using mesenchymal stem cells (MSCs) seems to be the most promising approach to heal tendon. Moreover, providing safe and regulated cell therapy products to patients requires adherence to good manufacturing practices (GMP). Adipose-derived stem cells (n=4) were cultured in 6-well plates coated with type-I collagen in a chemically defined serum-free medium (SF) or a xenogenic-free human pooled platelet lysate medium (hPL). At passage 4, ASCs were induced to tendon lineage for 14 days using 100ng/ml CTGF, 10ng/ml TGFβ3, 50ng/ml BMP12 and 50µg/ml ascorbic acid in the SF (SF-TENO) or in the hPL (hPL-TENO) medium. Cells cultured without any supplements are used as control. Morphological appearance, cell viability and FACS were performed in undifferentiated cells to evaluate the xenogenic-free culture conditions; the gene and protein expression were performed by RT-PCR and immunofluorescence to evaluate to expression of stem cell- and tendon-related markers upon cell differentiation. SF-CTRL and hPL-CTRL showed similar viability and MSC's surface proteins and expressed the stemness markers NANOG, OCT4 and Ki67. Moreover, both SF-TENO and hPL-TENO expressed significant higher levels of SCX, COL1A1, COL3A1, COMP, MMP3 and MMP13 genes already at 3d (p<0.05) respect to CTRLs. Scleraxis and collagen were also detected in both SF-TENO and hPL-TENO at protein level in higher amount than CTRLs. In conclusion, ASCs exposed to CTGF, BMP12, TGFb3 and AA in both serum and xenogenic-free media possess similar tenogenic differentiation ability moving forward the GMP-compliant approaches for the clinical use of ASCs

Introduction: Seven patients underwent successful revision total knee replacement for aseptic loosening. Bovine bone graft was used to reconstruct bony defects in all. Materials and methods: This is a retrospective review. Between April 2000 and March 2003, bovine bone (Tutobone™, Wescott-Medical, UK) was used in 7 revision arthroplasty cases (4 right knees & 3 left). There were 5 males and 2 females. The average age was 70.4 years. All revisions were carried out for aseptic loosening of the prostheses associated with massive osteolysis and bone loss. The bone defects on the tibia and femur were as follows: (Obtained from operative records. Classified according to Anderson Orthopaedic Research Institute classification). Type I. Type IIA. Type IIB. Type III. TIBIA. 3. 1. 2. 1. FEMUR. 2. 3. 2. 0. The tibial defects were corrected by impaction grafting and femoral condyle defects were corrected by using bovine bone as bulk grafts. Semi-constrained constrained stemmed cemented modular knee prostheses (TC3, Depuy) were used in all. Clinical outcomes were recorded by the Oxford Knee Score. Serial radiographs were evaluated for graft density, integration, implant loosening, alignment and subsidence. Results: At recent follow-up, radiographs showed good graft integration, no loosening, and no subsidence of the implant and good prostheses alignment. The average Oxford Knee Score was 20.4. Conclusion & discussion: Bovine bone substitute is an alternative. The bone defects in these patients were successfully reconstructed with bovine bone. It is an osteo-conductive matrix with intact type-I collagen that provides mechanical stability. It is also cost effective. Early results are encouraging but long-tem follow-up is needed

We aimed to determine whether development of heterotopic ossification (HO) following THA might be predicted by early changes in biochemical markers of bone turnover. The study cohort consisted of 21 men and women taking part in a randomised trial of the bisphosphonate pamidronate in the prevention of bone loss following THA. All had under gone unilateral THA using the same design of implant and all were assigned to placebo in the trial. The osteoblast activity markers bone-specific alkaline phosphatase (bAP), osteocalcin (Oc), and N-terminal propeptide of type-I procollagen (PINP); and the osteoclast activity markers deoxypyridinoline (iFDpd) and N-telopeptide of type-I collagen (NTx) were measured at baseline, and at 1, 6, 12, and 26 weeks following unilateral THA. The presence of HO was assessed using the Brooker grading by a musculoskeletal radiologist from plain AP radiographs of the hip taken at week 26. A transient increase in all turnover markers occurred following surgery, with peaks in iFDpd, NTx, and PINP at 6 weeks, and peaks in bAP and Oc at 12 weeks. 10 subjects had HO at week 26 (all Brooker grade 1 or 2). Subjects with HO had higher mean peak rises (SEM) in PINP and Oc than those without HO (PINP 81% (10) versus 43% (10), P=0.01; Oc 26% (5) versus 9% (6), P=0.04). Using area under the curve ‘ROC’ analysis, PINP and Oc were equally discriminatory in predicting HO formation (P< 0.05). The optimal cut-off peak rise of > 57% in PINP at 6 weeks following THA had a sensitivity and specificity of 90 and 82, respectively for predicting the development of HO. An increase in PINP of more than 57% 6 weeks following THA is predictive of the development of HO at 26 weeks. This early prediction might allow identification of patients in whom early therapeutic measures could be taken

The material most widely used in orthopaedics is hydroxyapatite (HA), anyway many differences are still present between synthetic HA and biological HA. The aim of this study was to compare adhesion, proliferation and differentiation of human osteoblast-like cells on hydroxyapatite discs with different porosity and on plastic cultures. Human osteoblast-like cells were isolated from 4 young patients (mean age 24.5 years old), treated with collagenase and maintained in Dulbecco’s modified essential medium-10% fetal calf serum. Cells were plated on hydroxyapatite discs with 3 different porosities (35%, 35–55% e 55%) and on plastic cultures used as control. The proliferation was determined by the MTT colorimetric method, and alkaline phosphatase (ALP) activity was measured by a spettrophotometric method. Type I collagen and osteonectin production were demonstrated with fluorescence microscopy and osteoblast adhesion was studied by scanning electron microscopic (SEM) analysis. Results were analysed by one-way analysis of variance (ANOVA). Osteoblast proliferation on HA was three- to six-fold lower then on plastic. At 28 days, 2141 (± 350) cells/well grew on the most porous disks, with highly significant differences from controls. The ALP production was 2–3 fold lower on HA than on plastic. In the most porous disks, the mean ALP activity was of 2.95 (± 0.07) UI/well after 28 days, higher than in the other two groups. The type-I collagen and the osteonectin fluorescence reaction evidenced a cytoplasmic and a matrix labeling on HA at different porosities. SEM analysis showed osteoblasts with a flattened morphology and only few of them were metabolic active. At 21 and 28 days, proliferation rate and ALP activity on the three HA cultures were significantly different (p< 0.05). A decrease in cell population and increased ALP activity were observed on the most porous material, and high proliferation and poor differentiation rates on the less porous disks

Charcot neuroarthropathy is a rare but serious complication of diabetes, causing progressive destruction of the bones and joints of the foot leading to deformity, altered biomechanics and an increased risk of ulceration.

Management is complicated by a lack of consensus on diagnostic criteria and an incomplete understanding of the pathogenesis. In this review, we consider recent insights into the development of Charcot neuroarthropathy.

It is likely to be dependent on several interrelated factors which may include a genetic pre-disposition in combination with diabetic neuropathy. This leads to decreased neuropeptides (nitric oxide and calcitonin gene-related peptide), which may affect the normal coupling of bone formation and resorption, and increased levels of Receptor activator of nuclear factor kappa-B ligand, potentiating osteoclastogenesis.

Repetitive unrecognized trauma due to neuropathy increases levels of pro-inflammatory cytokines (interleukin-1β, interleukin-6, tumour necrosis factor α) which could also contribute to increased bone resorption, in combination with a pre-inflammatory state, with increased autoimmune reactivity and a profile of monocytes primed to transform into osteoclasts - cluster of differentiation 14 (CD14).

Increased blood glucose and loss of circulating Receptor for Advanced Glycation End-Products (AGLEPs), leading to increased non-enzymatic glycation of collagen and accumulation of AGLEPs in the tissues of the foot, may also contribute to the pathological process.

An understanding of the relative contributions of each of these mechanisms and a final common pathway for the development of Charcot neuroarthropathy are still lacking.

Cite this article: S. E. Johnson-Lynn, A. W. McCaskie, A. P. Coll, A. H. N. Robinson. Neuroarthropathy in diabetes: pathogenesis of Charcot arthropathy. Bone Joint Res 2018;7:373–378. DOI: 10.1302/2046-3758.75.BJR-2017-0334.R1.

Objectives

Matrix-assisted autologous chondrocyte transplantation (MACT) has been developed and applied in the clinical practice in the last decade to overcome most of the disadvantages of the first generation procedures. The purpose of this systematic review is to document and analyse the available literature on the results of MACT in the treatment of chondral and osteochondral lesions of the knee.