Monolayer expansion of human articular chondrocytes (HAC) is known to result in progressive dedifferentiation and loss of stable cartilage formation capacity in vivo. For optimal outcome of chondrocyte based repair strategies, HAC capable of ectopic cartilage formation may be required. Thus, the aim of this study was to establish appropriate quality control measures capable to predict the ectopic cartilage formation capacity of HAC from culture supernatants. This strategy would avoid the waste of cells for quality control purposes, in order to improve cell therapy and tissue-engineering approaches for the repair of joint surface lesions. Standardized medium supernatants (n=5) of freshly isolated HAC and chondrocytes expanded for 2 (PD2) or 6 population doublings (PD6) were screened for 15 distinct interleukins, 8 MMPs and 11 miscellaneous soluble factors by a multiplexed immunoassay. Cartilage differentiation markers like COMP and YKL-40 were determined by ELISA. Corresponding HAC were subcutaneously transplanted into SCID-mice and their capacity to form stable ectopic cartilage was examined histologically 4 weeks later. While freshly isolated chondrocytes generated stable ectopic cartilage positive for collagen type II, none of the PD6 transplants formed cartilaginous matrix. Loss of ectopic stable cartilage formation capacity between PD0 and PD6 correlated with a drop of MMP3 secretion to < 10% of initial levels, while changes for other investigated molecules were not predictive. Chondrocytes from donors with low MMP3 levels (< 10%) at PD2 failed to regenerate ectopic cartilage at PD2, indicating that MMP3 levels of cultured chondrocytes, independent of the number of cell doublings and the time in culture, predicted ectopic cartilage formation. In conclusion, loss of stable ectopic cartilage formation capacity in the course of HAC dedifferentiation can be predicted by determination of relative MMP3 levels demonstrating that standardized culture supernatants can be used for quality control of chondrocytes dedicated for cell therapeutic approaches

Summary Statement. This work raises the potential of utilizing stem cells to catalyze cartilage regeneration by a minimal number of neonatal chondrocytes via controlling cell distribution in 3D matrices, and may solve the challenge of scarce donor availability associated with cell-based therapy. Introduction. Cartilage loss is a leading cause of disability among adults and represents a huge socio-economical burden. Allogeneic neonatal articular chondrocytes (NChons) is a promising cell source for cartilage regeneration because these cells are highly proliferative, immune-privileged, and readily produce abundant cartilage matrix. However, scarce donor availability for NChons greatly hinders their broad clinical application. Besides their ability to differentiate into different tissue types, stem cells may contribute to tissue regeneration through the secretion of paracrine factors. Here we examined the potential for using a minimal number of NChons to catalyze cartilage tissue formation by co-culturing them with adipose-derived stem cells (ADSCs) in 3D biomimetic hydrogels. Materials & Methods. NChons were isolated from articular cartilage of a three-day old calf. Human adult ADSCs were expanded to passage 5. Cells were photo-encapsulated in a hydrogel consisting of 7% w/v poly(ethylene glycol diacrylate) and 3% w/v chondroitin sulfate-methacrylate. To examine the effects of different paracrine concentrations, NChons and ADSCs were co-cultured in three different co-culture models: 1) cells cultured with conditioned medium supplementation from the other cell type (CM), 2) bi-layered co-culture confining each cell type to its own layer (BI), and 3) mixed cell co-culture at different ratios (75C:25A, 50C:50A, 25C:75A, 10C:90A). Cell-hydrogel constructs were cultured for 3 weeks in chondrogenic medium with 10ng/ml TGF-β3 and analyzed for biochemical content (DNA, sulfated glycosaminoglycan (sGAG), and collagen) and immunostaining. Fluorescent cell membrane labeling was used to identify ADSCs in mixed co-culture. To quantify interaction synergy, the interaction index, defined as the measured biochemical content in the mixed co-culture normalised by the expected value based on cell ratio and the measured content in the controls, was calculated (2). Statistical significance (∗) was set to p<0.05. Results. At day 21, mixed co-culture with as low as 25% NChons led to higher cell number and cartilage matrix content than NChon control. ADSC control had significantly lower matrix content. In mixed co-culture, the interaction index for DNA, sGAG, and collagen increased with an increase in ADSC ratio, reaching up to 5–6 at 90% ADSCs. Immunostaining of collagen II revealed that mixed co-culture resulted in the formation of large cartilage nodules, and that nodule size increased with an increase ADSC ratio. Cell tracking showed that the labeled ADSCs always resided outside the cartilage noduless, indicating the cartilage nodules are composed entirely of NChons. Discussion & Conclusion. In this study, we demonstrated the efficacy of harnessing the paracrine effects ADSCs to catalyze cartilage tissue formation by a small number of NChons in biomimetic hydrogels. The mild effects of CM and BI co-culture on cartilage tissue formation along with the increase in interaction synergy with ADSC ratio in mixed co-culture highlighted the importance of using 3D scaffolds to probe cell-cell interactions in a spatially controlled manner. Such strategy significantly reduces the number of NChons needed, which may accelerate the translation of NChon for cartilage repair by alleviating donor scarcity limitation, and may be broadly applicable to regenerating other tissue types

In the native articular cartilage microenvironment, chondrocytes are constantly subjected to dynamic physical stimuli that maintains tissue homeostasis. They produce extra cellular matrix (ECM) components such as collagens (type II mainly, 50-75%), proteoglycans (10-30%) and other type of proteins. 1. . While collagen offers a large resistance in tension, proteoglycans are the responsible of the viscoelastic response under compression due to the negative charge they confer to the ECM allowing it to entrap a large amount of interstitial fluid. In pathologic states (e.g. osteoarthritis), this ECM is degenerated and the negative charge becomes unbalanced, losing the chondroprotective properties and resulting on an overloaded chondrocytes that further degenerate the matrix. Low-Intensity Pulsed Ultrasound Stimulation (LIPUS) has been used to generate acoustic (pressure) waves that create bubbles that collapse with cells, inducing a stimulus that can modulate cell response. 2. This mechanical stimulation promotes the expression of type II collagen, type X collagen, aggrecan and TGF-β, appearing as a great strategy to regenerate cartilage. However, current strategies make use of extrinsic forces to stimulate cartilage formation overlooking the physico-chemical properties of the degenerated cartilage, resulting in an excessive load-transfer to chondrocytes and the consequent hypertrophy and degeneration. Here, interpenetrated networks (IPNs) with different compositions were created using methacrylated gelatin (GelMA), to mimic the collagen, and alginate functionalized with tyramine (Alg-tyr) to mimic glycosaminoglycans and to introduce a negative charge in the model. Within the matrix chondrocytes where encapsulated and stimulated under different conditions to identify the ultrasound parameters that enhance tissue formation. Samples with and without stimulation were compared analysing the expression and deposition of collagen II, aggrecan, collagen X and TGF-β. The results suggested that the chondrogenic marker expression of the samples stimulated for 10 minutes per day for 28 days, was two times higher overall in all of the cases, which was correlated to the tissue formation detected. Acknowledgments: The authors would like to thank the Basque Government for the “Predoctoral Training Program for Non-Doctoral Research Staff 2021-2022” (Grant ref.: PRE_2021_1_0403). This work was supported by the RETOS grant PID2020-114901RA-I00 of the Ministry of Science and Innovation (MICINN)

Cartilage is a realistic target for tissue engineering given the avascular nature and cellular composition of the tissue. Much of the work in this field has been largely empirical, indicating the need for alternative approaches to the design of cartilage formation protocols. Given the heterogeneity associated with human mesenchymal populations, continuous cell lines may offer an alternative to model and simplify cartilage generation protocols. We therefore exploited the potential of the murine chondrocytic ATDC5 cell line to, i) delineate the process of chondrocyte differentiation in monolayer culture and three-dimensional micromass pellet culture systems, and ii) model cartilage formation utilising appropriate scaffold and bioreactor (perfused and rotating) technologies. Monolayer cultures of ATDC5 cells over a 28-day period in presence of insulin demonstrated various stages of chondrocyte differentiation- proliferative, pre-hypertrophic, hypertrophic and finally, mineralisation of cartilaginous nodules. This was confirmed by gene and protein expression, by qPCR and Western blotting respectively, of chondrogenic differentiation markers- Sox-9, Bcl-2, Type II and X collagens. Pellet cultures of ATDC5 cells under chondrogenic conditions (10 ng/ml TGF-beta3, 1X ITS {insulin, transferrin, selenium}, 10 nanomolar dexamethasone, 100 micromolar ascorbate-2-phosphate) illustrated a gradual progression from an aggregation of cells at day 7, to initiation of matrix synthesis at day 14, followed by formation of well-defined cartilaginous structures at day 21. Chondrogenic differentiation at day 21 was evident by numerous proliferative/ pre-hypertrophic chondrocytes, staining for Sox-9, Aggrecan, Type II collagen and PCNA, lodged in distinct lacunae embedded in cartilaginous matrix of proteogly-cans and Type II collagen. Inclusion of TGF-beta3 in the chondrogenic medium during pellet culture beyond 21 days maintained the pre-hypertrophic phenotype, even at day 28. In contrast, removal of TGF-beta3, addition of 50 nanomolar thyroxine and reduction of dexa-methasone to 1 nanomolar in the chondrogenic medium stimulated hypertrophy at day 28, evident by down-regulation of Sox-9 expression. ATDC5 cells cultured on Polyglycolic acid fleece in the rotating bioreactor or encapsulated in chitosan /alginate and cultured in the perfused bioreactor for 21 days, formed cartilaginous explants reminiscent of hyaline cartilage. Thus, ATDC5 cells constitute an ideal cell line to elucidate the steps of chondrocyte differentiation and cartilage formation

In recent years numerous growth factors acting on musculoskeletal tissues have been identified. This presentation summarizes our experience with IGF1 in the stimulation of growth of the physis and TGF beta in the formation of bone and cartilage. IGF1 in a carrier, agarose, was instilled in a paraphsyeal region in rabbit tibias. The physeal height was measured over a period of time and was found to have increased in the group treated with IGF1 when compared to the control group. In addition there was delayed closure of the physeal plate. These findings may have clinical applications in stimulation of physeal growth in small by length discrepancies. A polycaprolactone scaffold impregnated with TGF beta was implanted under the skin, in the muscle and under the periosteum in rabbits. Over a period of time the scaffolds were harvested and subjected to histological analysis with a variety to stains. Formation of bone and cartilage was found in these scaffolds implanted under the periosteum. Subdermal and intramuscular implantation of the scaffolds did not produce the same results. It is postulated that apart from TGF Beta local and environmental factors may play a part in bone and cartilage formation. This model may be useful in creating complex scaffolds in-vivo for subsequent transplantations

Osteoarthritis (OA) is a chronic degenerative joint disease characterized by progressive cartilage degradation, synovial membrane inflammation, osteophyte formation, and subchondral bone sclerosis. Pathological changes in cartilage and subchondral bone are the main processes in OA. In recent decades, many studies have demonstrated that activin-like kinase 3 (ALK3), a bone morphogenetic protein receptor, is essential for cartilage formation, osteogenesis, and postnatal skeletal development. Although the role of bone morphogenetic protein (BMP) signalling in articular cartilage and bone has been extensively studied, many new discoveries have been made in recent years around ALK3 targets in articular cartilage, subchondral bone, and the interaction between the two, broadening the original knowledge of the relationship between ALK3 and OA. In this review, we focus on the roles of ALK3 in OA, including cartilage and subchondral bone and related cells. It may be helpful to seek more efficient drugs or treatments for OA based on ALK3 signalling in future

Osteoarthritis is a degenerative disease that results in changes in cartilage extracellular matrix. In vitro studies have shown that IL-1β inhibits cartilage formation in chondrocytes or MSCs undergoing chondrogenesis. In vivo, articular chondrocytes and bone marrow reside under hypoxic or physioxic environment (1–5% oxygen) and previous investigations have shown an increase in cartilage matrix proteins and reduced hypertrophy for MSC chondrogenesis, especially for MSCs expanded and differentiated under physioxia. Our hypothesis was that physioxic preconditioning reduces the effects of IL-1β inhibited MSC chondrogenesis. Methods. Human MSCs (Male donors; aged 18–60 years, n = 6) were isolated from bone marrow and expanded for one passage and split into hyperoxic and physioxic MSC cultures, the latter conditions were isolated and expanded using a hypoxia controlled incubator. MSCs with or without physioxic preconditioning were aliquoted into wells of a 96-well cell culture plate in the presence of 10ng/ml TGF-β. 1. or in combination with either 0.1 or 0.5ng/ml IL-1ß and centrifuged to form pellets. Pellets were then differentiated under their isolation conditions. Pellets removed from culture on days 7, 14 and 21, were evaluated for wet weight, histological (DMMB staining, collagen type I, II, MMP-13 and TGF-β receptor II) and collagen type II ELISA analysis. Results. Preconditioned MSCs demonstrated an enhanced collagen type II and GAG production undergoing chondrogenesis compared to hyperoxic pellets. In the presence of IL-1β, preconditioned MSCs reduced the inhibitory effect of IL-1ß compared to the equivalent conditions under hyperoxic, whereby there was a significant increase in wet weight, GAG and collagen type II production (p < 0.05). Furthermore, preconditioning MSCs had reduced collagen type X expression compared to hyperoxic cultures. Discussion. Preconditioned MSCs had enhanced matrix formation compared to hyperoxic cultures. In the presence of IL-1ß, preconditioned MSCs and physioxic differentiation reduced the inhibitory effects of IL-1ß. This may be related to restoration of TGF-ß receptor II expression (anabolic effect) and reduced expression of catabolic enzyme, MMP-13. The latter enzyme is also involved in hypertrophy and so physioxia helps to partially restore articular cartilage phenotype

Problems of vertebral growth plate metabolism regulation at different stages of ontogenesis are insufficiently covered in the literature. However, the study of function mechanism of provisional cartilage of vertebral growth plate is a practical and theoretical basis of pathogenesis model of idiopathic scoliosis and Scheuermann’s disease both associated with growth disorders.

Objective: To investigate the function mechanism of vertebral growth plate structural components during formation and growth.

Materials and methods: Fifty vertebral body specimens of children at the age from 1 to 14 years obtained from the forensic medicine department were studied by methods of morphohistochemistry, biochemistry, and ultra-structural analysis. The expression of five proteoglycan genes and their albuminous products was investigated by RT-PCR method.

Results: The process of growth represents a sequence of morphogenetic movements ongoing up to the achievement of sexual maturity. But morphofunctional organization and regulation of growth are different in different periods of ontogenesis. Early postnatal growth of vertebral bodies is governed by a radially located zone of growth. The cell population in a just-formed cartilage growth plate is non-uniform: from poorly differentiated chondroblast through the form of highly differentiated ones to degrading chondrocyte. This period of the spine development is characterised by the presence of vessels in provisional cartilage tissue. The concept of “chondro/hematic barrier” suggested and validated by A.M Zaidman explains a conservation of homeostasis at a stage of vertebral bodies differentiation. The process of chondrogenic differentiation of prechondroblasts in the early postnatal period is inducted by the chorda influence. In the late postnatal period (12–14 years) the laws of structural and functional organization of cartilage growth plate of vertebral body remain the same: phenotypic heterogeneity, polarity, and zonality of cells. A metabolic centre of complex architectonics of cartilage tissue is chondroblast. Chondroblast is functioning at the level of chondron which is a functional unit of vertebral growth plate. Chondroblast (chondrocyte) is located in the centre of chondron and surrounded by pericellular matrix presented by diffuse aggrecan molecules, or growth plate aggregates.

Due a peculiar architectonics, growth plate molecules have inner spaces comparable in size with Golgi’s vesicles. Metabolites, small molecules, and water freely penetrate through these molecules. Diffuse molecules together with type II thin collagenic fibres, minor collagenes, and structure-forming growth plates perform barrier function. Besides barrier function, diffuse molecules perform information function inside a chondron, forming a kind of information field. Signals of this field are perceived by chondroblast receptors, and the cell gene apparatus expression is carried out through second messengers. Thus, either stimulation of proliferative activity with subsequent differentiation during intensive growth, or interruption of these processes (period of growth delay) occurs. Single chondrons unite into chains in proliferation zones. Cell interaction inside chondron occurs due transmembrane structures, as a contact coordination of functions of cells with inherent high specificity. Concentration of diffuse molecules of growth plate (aggrecan) in proliferation zones is the highest on evidence of histochemical and ultrastructural assays. Besides, diffuse molecules are the short-distance regulators of DNA synthesis the mechanism of action of which is realised through the system of receptors on a cellular membrane. Hence, contact intercellular interactions are one of the mechanisms controlling cell division. These are so-called extracellular factors of chondroblast proliferation regulation.

Thus, the process of growth represents a complex two-stage mechanism of proliferation and differentiation of chondroblasts, and adequate osteogenesis. All three processes provide harmonious spine formation, and disturbance of one of them results in pathology development.

Herein we address, hyaline cartilage regeneration issue by engineering a synthetic biocompatible hydrogel scaffold capable to promote chondrogenic differentiation. In this study, the chemically crosslinked hydrogels consisting of synthetic peptides that have the collagen-like sequence Cys-Gly-(Pro-Lys-Gly)4 (Pro-Hyp-Gly)4 (Asp-Hyp-Gly)4- conjugated with RGD sequence (CLP-RGD) and crosslinked hydrogels of type I collagen (CA) were used. For cartilage formation, we used human skeletal muscle-derived stem/progenitor cells (hMDSPCs) set for differentiation towards a chondrogenic lineage by BMP-7 and TGF-ß3 growth factors. Initially 150, 100 and 75 ng of BMP-7and TGF-ß3 growth factors were inserted in each scaffold and amount of growth factors diffusing out of the scaffolds was observed by ELISA assays. In vitro experiments were performed by seeding hMDSPCs onto hydrogels loaded with growth factors (75ng/scaffold) and cultured for 28 days. Cartilage formation was monitored by ELISA and RT-PCR assays. All experiments were performed in triplicates or quadruplicates. Growth factors incorporation strategy allowed a sustained release of TGF-ß3 growth factor, 6.00.3% of the initially loaded amount diffused out after 4 h and 2.70.5% already at the second time point (24h) from CA and CLP-RGD substrates. For the BMP-7 growth factor, 13.12.3% and 15.751.6% of the initially loaded amount diffused out after 4 h, 1.70.2% and 2.450.3% at the second time point (24 h) from CA and CLP-RGD respectively. In vitro experiments shown that scaffolds with immobilized growth factors resulted in higher collagen type II accumulation when compared to the scaffolds alone. The gene expression on CLP-RGD hydrogels with growth factors has shown lower collagen type I expression and higher aggrecan expression compared to day 0. However, we also report increased collagen X gene expression on CA hydrogels (with growth factors). Our results support the potential of the strategy of combining hydrogels functionalized with differentiation factors toward improving cartilage repair

Aims. Extracellular matrix (ECM) and its architecture have a vital role in articular cartilage (AC) structure and function. We hypothesized that a multi-layered chitosan-gelatin (CG) scaffold that resembles ECM, as well as native collagen architecture of AC, will achieve superior chondrogenesis and AC regeneration. We also compared its in vitro and in vivo outcomes with randomly aligned CG scaffold. Methods. Rabbit bone marrow mesenchymal stem cells (MSCs) were differentiated into the chondrogenic lineage on scaffolds. Quality of in vitro regenerated cartilage was assessed by cell viability, growth, matrix synthesis, and differentiation. Bilateral osteochondral defects were created in 15 four-month-old male New Zealand white rabbits and segregated into three treatment groups with five in each. The groups were: 1) untreated and allogeneic chondrocytes; 2) multi-layered scaffold with and without cells; and 3) randomly aligned scaffold with and without cells. After four months of follow-up, the outcome was assessed using histology and immunostaining. Results. In vitro testing showed that the secreted ECM oriented itself along the fibre in multi-layered scaffolds. Both types of CG scaffolds supported cell viability, growth, and matrix synthesis. In vitro chondrogenesis on scaffold showed an around 400-fold increase in collagen type 2 (COL2A1) expression in both CG scaffolds, but the total glycosaminoglycan (GAG)/DNA deposition was 1.39-fold higher in the multi-layered scaffold than the randomly aligned scaffold. In vivo cartilage formation occurred in both multi-layered and randomly aligned scaffolds treated with and without cells, and was shown to be of hyaline phenotype on immunostaining. The defects treated with multi-layered + cells, however, showed significantly thicker cartilage formation than the randomly aligned scaffold. Conclusion. We demonstrated that MSCs loaded CG scaffold with multi-layered zonal architecture promoted superior hyaline AC regeneration. Cite this article: Bone Joint Res 2020;9(9):601–612

Patients with bone and muscle weakness from disuse have higher risk of fracture and worse post-injury mortality rates. The goal of this current study was to better inform post-fracture rehabilitation strategies by investigating if physical remobilization following disuse by hindlimb unloading improves osteochondral callus formation compared to continued disuse by hindlimb suspension (HLS). We hypothesized that continued HLS would impair callus bone and cartilage formation and that physical rehabilitation after HLS would increase callus properties. All animal procedures were approved by the VCU IACUC. Skeletally mature, male and female C57BL/6J mice (18 weeks) underwent HLS for 3 weeks. Mice then had their right femur fractured by open surgical dissection (stabilized with 24-gauge pin). Mice were then either randomly assigned to continued HLS or allow normal physical weight-bearing remobilization (HLS + R). Mice allowed normal cage activity throughout the experiment served as controls (GC). All mice were sacrificed 14-days following fracture with 4-8 mice (male and female) per treatment. Data analyzed by respective ANOVA with Tukey post-hoc (*p< 0.05; # p < 0.10). Male and female mice showed conserved and significant decreases in hindlimb callus bone formation from continued HLS versus HLS + R. Combining treatment groups regardless of mouse sex, histological analyses using staining on these same calluses demonstrated that HLS resulted in trends toward decreased cartilage cross-sectional area and increased osteoclast density in woven bone versus physically rehabilitated mice. In support of our hypothesis, physical remobilization increases callus bone formation following fracture compared to continued disuse potentially due to increased endochondral ossification and decreased bone resorption. In all, partial weight-bearing exercise immediately following fracture may improve callus healing compared to delayed rehabilitation regimens that are frequently used

Osteoarthritis is the most common chronic condition of the joints. It is characterized by the degeneration of articular cartilage, formation of osteophytes and alterations in the synovium. This process has a severe impact on the quality of life of the patients and the currently available treatments are unsatisfactory and often merely focused on pain relief. In our group we are working on the development of in situ cross-linkable hydrogel platforms that could be used for resurfacing the damaged articular cartilage using a minimally invasive arthroscopic procedure. Stable fixation of the gel at the joint surface, facilitating the ingrowth of local stem and progenitor cell populations and supporting intrinsic repair mechanisms are considered minimal design parameters. To achieve this, we are exploring the use of enzymatically cross-linkable natural polymer-tyramine conjugates. Dextran-tyramine conjugates were prepared by activation of dextran-OH and subsequent reaction with tyramine. Hyaluronic acid-tyramine and protein-tyramine conjugates were prepared using DMTMM coupling. In situ crosslinking is achieved by mixing the polymer conjugates with the enzyme HRP and minute, non-toxic amounts of H2O2 as oxidizing agent. Support of cartilage formation was studied after mixing of the polymer conjugates with mesenchymal stem cells, chondrocytes or combinations of both prior to crosslinking. Cell ingrowth was studied by implanting the hydrogels in an ex-vivo cartilage defect while mechanically loading the explant in a bioreactor and cell migration in the hydrogels was evaluated by tracking the sprouting of fluorescently labelled cell-spheroids. We prepared dextran-tyramine conjugates with a degree of substitution of 10 tyramine residues per 100 monosaccharide units. The conjugated hyaluronic acid-tyramine had a degree of substitution of 10% of the carboxylic acid groups, while for the proteins the substitution was dependent on the protein type. Enzymatically crosslinked hydrogels, based on dextran and hyaluronic acid, with the addition of co-cross linkable proteins show excellent properties for application in the regeneration of damaged cartilage

Osteoarthritis (OA) is the most common joint disease, which is characterized by a progressive loss of proteoglycans and the destruction of extracellular matrix (ECM), leading to a loss of cartilage integrity and joint function. During OA development, chondrocytes alter ECM synthesis and change their gene expression profile including upregulation of hypertrophic markers known from the growth plate. Although physiological mechanical loading can support cartilage formation and maintenance, mechanical overload represents one major risk factor for OA development. To date, little is known on how an OA-like hypertrophic chondrocyte phenotype alters the response of cartilage tissue to mechanical loading. The aim of this study was to investigate whether a hypertrophic phenotype change of chondrocytes affects the response to physiological mechanical loading and to reveal differences compared to normal control cartilage. Cartilage replacement tissue was generated using human articular chondrocytes (normal control cartilage, n=3–5) or human mesenchymal stromal cells which develop a hypertrophic phenotype similar to the one observed in OA (OA cartilage model, n=3–6). Cells were seeded in a collagen type I/III carrier and attached to a beta-TCP bone replacement phase, building an osteochondral unit for simulation of natural conditions. After 21 and 35 days of chondrogenic (re)differentiation, a single physiological mechanical compression episode (1 Hz, 25 %, 3 h) was applied, imitating three hours of normal walking in ten-minute intervals. Proteoglycan and collagen synthesis, gene expression and activation of signaling pathways were assessed. Cartilage replacement tissue of both groups had similar proteoglycan and collagen type II content as well as hardness properties. During (re)differentiation, both cell types showed a comparable upregulation of the chondrogenic marker genes COL2A1 and ACAN. As expected, hypertrophic marker genes (COL10A1, ALPL, MEF2C, IBSP) were only upregulated in the OA cartilage model. Mechanotransduction in both tissues was confirmed by load-induced activation of pERK1/2 signaling. While the 3 h loading episode significantly increased proteoglycan synthesis in normal control cartilage at day 35, the same protocol resulted in a suppression of proteoglycan and collagen synthesis in the OA cartilage model, which was accompanied by a downregulation of COL2A1 gene expression. In addition, hypertrophic marker genes COL10A1, ALPL and IBSP were significantly reduced after loading. Along lower load-induced SOX9 mRNA and protein stimulation in the OA cartilage tissue, a weaker induction of mechanosensitive BMP2, BMP6, FOS and FOSB gene expression was observed. While stable cartilage showed anabolic effects after physiological loading, the hypertrophic chondrocytes reacted with a reduced extracellular matrix synthesis. This could be explained by a lower mechanoinduction of the BMP signaling cascade and insufficient SOX9 stimulation. Progressive OA development could thus be influenced by a reduced mechanocompetence of osteoarthritic chondrocytes

Cartilage injuries often represent irreversible tissue damage because cartilage has only a low ability to regenerate. Thus, cartilage loss results in permanent damage, which can become the starting point for osteoarthritis. In the past, bioactive glass scaffolds have been developed for bone replacement and some of these variants have also been colonized with chondrocytes. However, the hydroxylapaptite phase that is usually formed in bioglass scaffolds is not very suitable for cartilage formation (chondrogenesis). This interdisciplinary project was undertaken to develop a novel slowly degrading bioactive glass scaffold tailored for cartilage repair by resembling the native extracellular cartilage matrix (ECM) in structure and surface properties. When colonized with articular chondrocytes, the composition and topology of the scaffolds should support cell adherence, proliferation and ECM synthesis as a prerequisite for chondrogenesis in the scaffold. To study cell growth in the scaffold, the scaffolds were colonized with human mesenchymal stromal cells (hMSCs) and primary porcine articular chondrocytes (pACs) (27,777.8 cells per mm. 3. ) for 7 – 35 d in a rotatory device. Cell survival in the scaffold was determined by vitality assay. Scanning electron microscopy (SEM) visualized cell ultramorphology and direct interaction of hMSCs and pACs with the bioglass surface. Cell proliferation was detected by CyQuant assay. Subsequently, the production of sulphated glycosaminoglycans (sGAGs) typical for chondrogenic differentiation was depicted by Alcian blue staining and quantified by dimethylmethylene blue assay assay. Quantitative real-time polymerase chain reaction (QPCR) revealed gene expression of cartilage-specific aggrecan, Sox9, collagen type II and dedifferentiation-associated collagen type I. To demonstrate the ECM-protein synthesis of the cells, the production of collagen type II and type I was determined by immunolabelling. The bioactive glass scaffold remained stable over the whole observation time and allowed the survival of hMSCs and pACs for 35 days in culture. The SEM analyses revealed an intimate cell-biomaterial interaction for both cell types showing cell spreading, formation of numerous filopodia and ECM deposition. Both cell types revealed initial proliferation, decreasing after 14 days and becoming elevated again after 21 days. hMSCs formed cell clusters, whereas pACs showed an even distribution. Both cell types filled more and more the pores of the scaffold. The relative gene expression of cartilage-specific markers could be proven for hMSCs and pACs. Cell associated sGAGs deposition could be demonstrated by Alcian blue staining and sGAGs were elevated in the beginning and end of the culturing period. While the production of collagen type II could be observed with both cell types, the synthesis of aggrecan could not be detected in scaffolds seeded with hMSCs. hMSCs and pACs adhered, spread and survived on the novel bioactive glass scaffolds and exhibited a chondrocytic phenotype

Adult articular cartilage mechanical functionality is dependent on the unique zonal organization of its tissue. Current mesenchymal stem cell (MSC)-based treatment has resulted in sub-optimal cartilage repair, with inferior quality of cartilage generated from MSCs in terms of the biochemical content, zonal architecture and mechanical strength when compared to normal cartilage. The phenotype of cartilage derived from MSCs has been reported to be influenced by the microenvironmental biophysical cues, such as the surface topography and substrate stiffness. In this study, the effect of nano-topographic surfaces to direct MSC chondrogenic differentiation to chondrocytes of different phenotypes was investigated, and the application of these pre-differentiated cells for cartilage repair was explored. Specific nano-topographic patterns on the polymeric substrate were generated by nano-thermal imprinting on the PCL, PGA and PLA surfaces respectively. Human bone marrow MSCs seeded on these surfaces were subjected to chondrogenic differentiation and the phenotypic outcome of the differentiated cells was analyzed by real time PCR, matrix quantification and immunohistological staining. The influence of substrate stiffness of the nano-topographic patterns on MSC chondrogenesis was further evaluated. The ability of these pre-differentiated MSCs on different nano-topographic surfaces to form zonal cartilage was verified in in vitro 3D hydrogel culture. These pre-differentiated cells were then implanted as bilayered hydrogel constructs composed of superficial zone-like chondro-progenitors overlaying the middle/deep zone-like chondro-progenitors, was compared to undifferentiated MSCs and non-specifically pre-differentiated MSCs in a osteochondral defect rabbit model. Nano-topographical patterns triggered MSC morphology and cytoskeletal structure changes, and cellular aggregation resulting in specific chondrogenic differentiation outcomes. MSC chondrogenesis on nano-pillar topography facilitated robust hyaline-like cartilage formation, while MSCs on nano-grill topography were induced to form fibro/superficial zone cartilage-like tissue. These phenotypic outcomes were further diversified and controlled by manipulation of the material stiffness. Hyaline cartilage with middle/deep zone cartilage characteristics was derived on softer nano-pillar surfaces, and superficial zone-like cartilage resulted on softer nano-grill surfaces. MSCs on stiffer nano-pillar and stiffer nano-grill resulted in mixed fibro/hyaline/hypertrophic cartilage and non-cartilage tissue, respectively. Further, the nano-topography pre-differentiated cells possessed phenotypic memory, forming phenotypically distinct cartilage in subsequent 3D hydrogel culture. Lastly, implantation of the bilayered hydrogel construct of superficial zone-like chondro-progenitors and middle/deep zone-like chondro-progenitors resulted in regeneration of phenotypically better cartilage tissue with higher mechanical function. Our results demonstrate the potential of nano-topographic cues, coupled with substrate stiffness, in guiding the differentiation of MSCs to chondrocytes of a specific phenotype. Implantation of these chondrocytes in a bilayered hydrogel construct yielded cartilage with more normal architecture and mechanical function. Our approach provides a potential translatable strategy for improved articular cartilage regeneration using MSCs

Recent advances in tissue engineering have made progress towards the development of biomaterials with the capability for delivery of growth factors to promote enhanced tissue repair. However, controlling the release of these growth factors is a major challenge and the associated high costs and side effects of uncontrolled delivery of has proved increasingly problematic in clinical orthopaedics. Gene therapy might be a valuable tool to avoid these limitations. While non-viral vectors are typically inefficient at transfecting cells, our group have had significant success in this area using a scaffold-mediated gene therapy approach for regenerative applications. These gene activated scaffold platforms not only act as a template for cell infiltration and tissue formation, but also as a ‘factory’ to provoke autologous host cells to take up specific genes and then engineer therapeutic proteins in a sustained but eventually transient fashion. Alternatively, scaffold-mediated delivery of siRNAs and miRNAs can be used to silence specific genes associated with pathological states in orthopaedics. This presentation will provide an overview of some of this research with a particular focus on gene-activated biomaterials for promoting stable cartilage formation in joint repair and on scaffold-based delivery of therapeutics for enhancing vascularization & bone repair

Tissue engineering techniques, combining autologous chondrocytes with biodegradable biomaterials, may offer significant advantages over current articular cartilage repair strategies. We present a series of experiments investigating the effect of 3D scaffold architecture and biomaterial composition on cartilage tissue formation in vitro and in vivo. Porous polymer (PEGT/PBT) scaffolds with low (300/55/45) or high (1000/70/30) PEG molecular weight (MW) compositions were produced using novel solid free-form fabrication (3DF) techniques, allowing precise control over pore architecture, and conventional compression moulding (CM) foam techniques. Scaffolds were seeded with expanded human nasal chondrocytes, and cultured in vitro or implanted subcutaneously in vivo in nude mice for 4 weeks and cartilage tissue formation accessed. 3DF scaffolds contained highly accessible networks of large interconnecting pores (Ø525 μm) compared to CM scaffolds, containing complex networks of small interconnecting pores (Ø182 μm). 3DF scaffold architectures enhanced cell re-differentiation (GAG/DNA) and cartilaginous matrix accumulation compared to CM scaffolds, but only if 1000/70/30 compositions were used. Collagen type-II mRNA was significantly increased in 3DF architectures irrespective of scaffold composition. These effects were likely mediated by preferential protein adsorption to 1000/70/30 materials, promoting a spherical chondrocyte-like morphology, as well as efficient nutrient/waste exchange throughout interconnecting pores within 3DF architectures. We observed synergistic effects of both composition and 3D scaffold architecture on human chondrocyte re-differentiation capacity, however, our data suggests that scaffold composition has a more significant influence than architecture alone. Such design criteria could be included in future scaffold architectures for repairing articular cartilage defects

Introduction Experimental heterotopic bone formation in the canine urinary bladder has been observed for more than seventy years without revealing the origin of the osteoinductive signals. In 1931, Huggins demonstrated bone formation in a fascial transplant to the urinary bladder. Through an elaborate set of experiments, it was found that proliferating canine transitional epithelial cells from the urinary system act as a source of osteoinduction. Urist performed a similar series of experiments in guinea pigs as Huggins did in his canine model. After two weeks, mesenchymal cells condensed against the columnar epithelium and membranous bone with haversian systems and marrow began to form juxtapose the basement membrane. At no time was cartilage formation noted, only direct membranous bone formation. They also demonstrated the expression of BMP’s in migrating epithelium and suggested that BMP is the osteoinductive factor in heterotopic bone formation. Method This study was approved by Institutional Animal Ethics Committee. Six dogs underwent a mid-line laparotomy incision followed by mobilisation of a right sided myoperitioneal vascularised flap based on an inferior epigastric artery pedicle. A sagittal cystotomy is made in the dome of the bladder and the vascularised flap was sutured in place with acryl absorbable, continuous suture. The animals were sacrificed at 6 weeks. The bladder samples were removed and assessed by histology and immunohistochemistry. Sections were incubated with optimal dilution of primary antibody for type I collagen, type III collagen, alkaline phosphatase (ALP), bone morphogenetic protein (BMP)-2 and –4, osteocalcin (OCN), osteopontin (OPN), bone sialoprotein (BSP). Results The mechanism for bone formation induced by the epithelial-mesenchymal cell interactions is not clear. We were able to demonstrate mature lamellar bone formation 6 weeks after transplanting a portion of the abdominal smooth muscle into the bladder wall. The bone formed immediately adjacent to the proliferating transitional uroepithelium, a prerequisite for bone formation in Huggins’ model. Here we report evidence of cartilage formation and therefore endochondral ossification as well as membranous bone formation. This is very similar histologically to the process of endochondral ossification at the growth plate in the growing skeleton. We propose a mechanism for the expression of BMP by epithelial cells. Discussion This study demonstrates transitional epithelium induced formation of chondrocytes and osteoblasts in muscle tissue. The sequential expression of bone matrix proteins was related to cell proliferation, differentiation and formation of endochondral and membranous bone. Further information regarding the molecular mechanism of bone formation induced by epithelial-mesenchymal cell interactions will improve understanding of cell differentiation during osteogenesis

Use of scaffolds for articular cartilage repair (ACR) has increased over the last years with many biomaterials options suggested for this purpose. It is known that scaffolds for ACR have to be optimally biodegradable with simultaneous promotion of chondrogenesis, favouring hyaline cartilage formation under rather complex biomechanical and physiological conditions. Whereas improvement of the scaffolds by their conditioning with stem cells or adult chondrocytes can be employed in bioreactors, “ideal” scaffolds should be capable of performing such functions directly after implantation. It was previously considered that scaffold structure and composition would be the best if it mimics the structure of native cartilage. However, in this case no clear reparative stimuli are being imposed on the scaffold area, which would drive chondrocytes activity in a desired way. In this work, we studied new xeno-free, recombinant human type III collagen-laden polylactide (PLA) mesh scaffolds, which have been designed, produced, and biomechanically optimized in vitro and in vivo validated in a porcine and equine model. The scaffolds were additionally assessed for relative performance simulated synovial fluids for both human conditions and veterinary cases. It was experimentally shown that success of the scaffolds in ACR eventually require lower stiffness than surrounding cartilage yet matching the strain compliance, different in static and dynamic conditions. This ensures an optimal combination of load transfer and oscillatory nutrients supply to the cells, which otherwise is difficult to rely on just with a passive diffusion in avascular cartilage conditions. The results encourage further development of such scaffold structures targeted on their best clinical performance rather than trying to imitate the respective original tissue. The authors would like to thank Finnish Agency for Innovation (Tekes) for providing financial support to this project. A.Z. also acknowledges Teknos Foundation (Finland) for the scholarship

Although osteochondral defects (OCD) following trauma, sport or degenerative diseases occur frequently, healing remains an unresolved clinical problem. These defects seem to appear more often in convex surfaces than in concave ones. In vivo studies have demonstrated the influence of mechanical conditions on osteochondral repair[. 1. ]. However, the influence of the local joint curvature on the mechanical environment as well as the effect of defect fillings on healing remained unknown. We hypothesize that healing of OCD is strongly affected by the local mechanical environment generated after variations in the joint geometry specifically on concave and convex joint surfaces. To study spontaneous repair, OCD (mm, 1.5mm depth) in 18 minipigs were created. Based on this knowledge, a predictive biphasic finite element model for tissue differentiation was created to simulate osteochondral healing. The model was validated by comparison of simulated healing with histological and histomorphometrical outcomes. Differentiation was regulated by the combination of a mechanical stimulus with a factor for differentiation defined for each tissue. The mechanical conditions arising from different predesigned defect fillings have been evaluated: Grafts with 100% (P1) and 50% (P2) of the native subchondral bone stiffness were analyzed. The healing pattern was in general qualitatively comparable to the findings of a gross examination of the histological sections. Generally, the pattern appears to be almost independent of the joint curvature. More hyaline cartilage (HC) was formed in the concave model during simulated healing. The maximum percentage of HC during the simulations was smaller and occurred earlier in the one (27 vs. 40%). In vivo 33% of HC was registered in the 12th week[. 2. ]. Defect filling restoring sub-chondral bone quality (P1) allowed a larger amount of hyaline cartilage formation than a less rigid filling (P2). Until today the more frequent occurrence of OCD at convex joint surfaces reported in the clinical practice has not been related to the local mechanical environment. This study is the first to demonstrate that this may be related to the mechanical stimulus for healing. In fact, during healing simulation HC formation was affected by changes in the joint surface curvature. A continuity of material properties in the layers under an OCD, which operates as basis for the newly formed cartilage, is important for the development of a tissue with adequate mechanical quality for load transmission. Indeed hyaline cartilage formation occurs earlier when P1 as when P2 was used. The use of a predictive tissue differentiation model allows a better understanding of the mechanical aspects of healing. Further analysis is however required before such algorithm may be applied in clinical cases. To consider mechanical factors affecting healing, appear to be of importance