Objectives. Bone tissue engineering is one of the fastest growing branches in modern bioscience. New methods are being developed to achieve higher grades of mineral deposition by osteogenically inducted mesenchymal stem cells. In addition to well established monolayer cell culture models, 3D cell cultures for stem cell-based osteogenic differentiation have become increasingly attractive to promote in vivo bone formation. One of the main problems of scaffold-based osteogenic cell cultures is the difficulty in quantifying the amount of newly produced extracellular mineral deposition, as a marker for new bone formation, without destroying the scaffold. In recent studies, we were able to show that . 99m. Tc-methylene diphosphonate (. 99m. Tc-MDP), a gamma radiation-emitting radionuclide, can successfully be applied as a reliable quantitative marker for mineral deposition as this tracer binds with high affinity to newly produced hydroxyapatite (HA). Methods. Within the present study, we evaluated whether this promising new method, using . 99m. Tc-hydroxydiphosphonate (. 99m. Tc-HDP), can be used to quantify the amount of newly formed extracellular HA in a 3D cell culture model. Highly porous collagen type II scaffolds were seeded with 1 × 106 human mesenchymal stem cells (hMSCs; n = 6) and cultured for 21 days in osteogenic media (group A – osteogenic (OSM) group) and in parallel in standard media (group B – negative control (CNTRL) group). After incubation with . 99m. Tc-HDP, the tracer uptake, reflected by the amount of emitted gamma counts, was measured. Results. We saw a higher uptake (up to 15-fold) of the tracer in the OSM group A compared with the CNTRL group B. Statistical analysis of the results (Student`s t-test) revealed a significantly higher amount of emitted gamma counts in the OSM group (p = 0.048). Qualitative and semi-quantitative analysis by Alizarin Red staining confirmed the presence of extracellular HA deposition in the OSM group. Conclusion. Our data indicate that . 99m. Tc-HDP labelling is a promising tool to track and quantify non-destructive local HA deposition in 3D stem cell cultures. Cite this article: T. L. Grossner, U. Haberkorn, T. Gotterbarm. . 99m. Tc-Hydroxydiphosphonate quantification of extracellular matrix mineralization in 3D human mesenchymal stem cell cultures. Bone Joint Res 2019;8:333–341. doi: 10.1302/2046-3758.87.BJR-2017-0248.R1

Aims. Here we introduce a wide and complex study comparing effects of growth factors used alone and in combinations on human mesenchymal stem cell (hMSC) proliferation and osteogenic differentiation. Certain ways of cell behaviour can be triggered by specific peptides – growth factors, influencing cell fate through surface cellular receptors. Methods. In our study transforming growth factor β (TGF-β), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), and vascular endothelial growth factor (VEGF) were used in order to induce osteogenesis and proliferation of hMSCs from bone marrow. These cells are naturally able to differentiate into various mesodermal cell lines. Effect of each factor itself is pretty well known. We designed experimental groups where two and more growth factors were combined. We supposed cumulative effect would appear when more growth factors with the same effect were combined. The cellular metabolism was evaluated using MTS assay and double-stranded DNA (dsDNA) amount using PicoGreen assay. Alkaline phosphatase (ALP) activity, as early osteogenesis marker, was observed. Phase contrast microscopy was used for cell morphology evaluation. Results. TGF-β and bFGF were shown to significantly enhance cell proliferation. VEGF and IGF-1 supported ALP activity. Light microscopy showed initial extracellular matrix mineralization after VEGF/IGF-1 supply. Conclusion. A combination of more than two growth factors did not support the cellular metabolism level and ALP activity even though the growth factor itself had a positive effect. This is probably caused by interplay of various messengers shared by more growth factor signalling cascades. Cite this article: Bone Joint Res 2020;9(7):412–420

Summary

45S5 bioactive glass combined with hMSC did not permit de novo ectopic bone formation. Such absence of osteogenicity was most likely due to the alkalinization of the 45S5 microenvironment that affects adversely the osteogenic differentiation of stem/precursor cells.

Bone marrow stromal cells (BMSCs) are capable of bone formation and can promote the repair of osseous defects when implanted in appropriate scaffolds. The most promising biomaterials for application in bone tissue engineering (TE) are hydroxyapatite (HA), tricalcium phosphate (TCP), calcium carbonate (coral) ceramics or bioactive glasses (BG) because of their osteoconductive properties and ability to enhance bone formation. However, information regarding the osteogenic potential of hBMSCs in combination with BG scaffolds is strikingly lacking in the TE field. The present study focused on evaluating the osteogenicity of bone constructs prepared from particles of 45S5 BG combined with hBMSCs in comparison with biphasic HA/TCP or coral particles, in a mouse ectopic model.

The in vivo osteogenicity was then correlated with various aspects of the effects of the scaffold materials tested on hBMSCs functions pertinent to bone tissue formation. Particular attention was given to the pH in the microenvironment where the cells reside in TE constructs and its effect on the osteoblastic differentiation of hBMSCs. In vivo experiments evidenced that 45S5 BG constructs with hBMSCs failed to form ectopic bone. In contrast, the cell constructs prepared with either HA/TCP or coral ceramics displayed great and consistent capacity for the ectopic bone formation. The cytocompatibility of hBMSCs on BG material was addressed and no differences were evidenced between HA/TCP and coral substrates related to the adhesion of hBMSCs and their proliferation in vitro. The hBMSCs viability was even higher within the 45S5 BG-containing constructs compared to the other two types of material constructs tested both in vitro and in vivo. These findings indicated that the absence of de novo bone formation in the hBMSCs-containing 45S5 BG constructs was not the result of cytotoxic effects of the BG material.

The potential of osteogenic differentiation of hBMSCs cultured on material substrates was next addressed and the ALP activity of hBMSCs was significantly diminished when these cells were cultured on 45S5 BG as compared to either HA/TCP or coral substrates. Because BG materials are well-known for causing external alkalinisation, the pH was specifically measured in TE constructs. The pH inside the cell-containing BG constructs, measured ex vivo, was 8.0 (i.e. 0.4–0.5 units more alkaline than that measured in the coral- or HA/TCP-constructs). The impact of such external alkalinisation on the osteogenic differentiation of hBMSCs was assessed by culturing the cells over a wide range of alkaline pH. The hBMSCs expression of osteogenic markers, ALP activity and mineralization were not significantly affected at moderate external alkaline pH (≤ 7.90) but were dramatically inhibited at higher pH.

Altogether, these findings provided evidence that despite 45S5 BG are reported to be good osteoconductive materials, they are not necessarily good scaffolds for TE, most likely due to the alkalinization of the 45S5 microenvironment that affects adversely the osteogenic differentiation of precursor cells. Controlling the shifts of pH in the local engineered extracellular environment is a critical issue for the development of bioactive TE scaffolds.

Aims. The involvement of long non-coding RNA (lncRNA) in bone marrow mesenchymal stem cell (MSC) osteogenic differentiation during osteoporosis (OP) development has attracted much attention. In this study, we aimed to disclose how LINC01089 functions in human mesenchymal stem cell (hMSC) osteogenic differentiation, and to study the mechanism by which LINC01089 regulates MSC osteogenesis. Methods. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) and western blotting were performed to analyze LINC01089, miR-1287-5p, and heat shock protein family A (HSP70) member 4 (HSPA4) expression. The osteogenic differentiation of MSCs was assessed through alkaline phosphatase (ALP) activity, alizarin red S (ARS) staining, and by measuring the levels of osteogenic gene marker expressions using commercial kits and RT-qPCR analysis. Cell proliferative capacity was evaluated via the Cell Counting Kit-8 (CCK-8) assay. The binding of miR-1287-5p with LINC01089 and HSPA4 was verified by performing dual-luciferase reporter and RNA immunoprecipitation (RIP) experiments. Results. LINC01089 expression was reinforced in serum samples of OP patients, but it gradually diminished while hMSCs underwent osteogenic differentiation. LINC01089 knockdown facilitated hMSC osteogenic differentiation. This was substantiated by: the increase in ALP activity; ALP, runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), and osteopontin (OPN) messenger RNA (mRNA) levels; and level of ARS staining. Meanwhile, LINC01089 upregulation resulted in the opposite effects. LINC01089 targeted miR-1287-5p, and the LINC01089 knockdown-induced hMSC osteogenic differentiation was repressed by miR-1287-5p depletion. HSPA4 is a downstream function molecule of the LINC01089/miR-1287-5p pathway; miR-1287-5p negatively modulated HSPA4 levels and attenuated its functional effects. Conclusion. LINC01089 negatively regulated hMSC osteogenic differentiation, at least in part, via governing miR-1287-5p/HSPA4 signalling. These findings provide new insights into hMSC osteogenesis and bone metabolism. Cite this article: Bone Joint Res 2024;13(12):779–789

Aims. Ageing-related incompetence becomes a major hurdle for the clinical translation of adult stem cells in the treatment of osteoarthritis (OA). This study aims to investigate the effect of stepwise preconditioning on cellular behaviours in human mesenchymal stem cells (hMSCs) from ageing patients, and to verify their therapeutic effect in an OA animal model. Methods. Mesenchymal stem cells (MSCs) were isolated from ageing patients and preconditioned with chondrogenic differentiation medium, followed by normal growth medium. Cellular assays including Bromodeoxyuridine / 5-bromo-2'-deoxyuridine (BrdU), quantitative polymerase chain reaction (q-PCR), β-Gal, Rosette forming, and histological staining were compared in the manipulated human mesenchymal stem cells (hM-MSCs) and their controls. The anterior cruciate ligament transection (ACLT) rabbit models were locally injected with two millions, four millions, or eight millions of hM-MSCs or phosphate-buffered saline (PBS). Osteoarthritis Research Society International (OARSI) scoring was performed to measure the pathological changes in the affected joints after staining. Micro-CT analysis was conducted to determine the microstructural changes in subchondral bone. Results. Stepwise preconditioning approach significantly enhanced the proliferation and chondrogenic potential of ageing hMSCs at early passage. Interestingly, remarkably lower immunogenicity and senescence was also found in hM-MSCs. Data from animal studies showed cartilage damage was retarded and subchondral bone remodelling was prevented by the treatment of preconditioned MSCs. The therapeutic effect depended on the number of cells applied to animals, with the best effect observed when treated with eight millions of hM-MSCs. Conclusion. This study demonstrated a reliable and feasible stepwise preconditioning strategy to improve the safety and efficacy of ageing MSCs for the prevention of OA development. Cite this article: Bone Joint Res 2021;10(1):10–21

While high-performance ceramics like alumina and zirconia exhibit excellent wear resistance, they provide poor osseointegration capacity. As osseointegration is crucial for non-cemented joint prostheses, new techniques have been successfully developed for biofunctionalizing high-performance ceramic surfaces. Stable cell adhesion can be achieved by covalently bound specific peptides. In this study we investigate the effect of sterilization processes on organo-chemically functionalized surfaces. To enhance the performance of alumina-toughened zirconia ceramics (ATZ), a 3-aminopropyldiisopropylethoxysilane (APDS) monolayer was applied and coupled with cyclo-RGD peptides (cRGD) by using bifunctional crosslinker bis(sulfosuccinimidyl)suberat (BS³). The samples were sterilized using e-beam or gamma-sterilization at 25 kGy, either before or after biofunctionalization with cRGD. Functionalization stability was investigated by contact angle measurements. The functionality of cRGD after sterilization was demonstrated using proliferation tests and cytotoxicity assays. Immunofluorescence staining (pFAK, Actin, DAPI) was conducted to evaluate the adhesion potential between the samples and human mesenchymal stem cells (hMSCs). Functionalized samples before and after sterilization showed no significant difference regarding their contact angles. A proliferation test demonstrated that the cells on functionalized samples proliferate significantly more than on untreated samples before and after sterilization. hMSCs showed a significant higher proliferation on gamma sterilized samples compared to all other groups after 14 days. It was confirmed that the samples did not exhibit cytotoxic behavior before or after sterilization. Fluorescence microscopy demonstrated that both, cells on sterilized and on non-sterilized samples, expressed high levels of pFAK-Y397. The investigated functionalization enables improved adhesion and proliferation of hMSCs and is stable against the investigated sterilization processes. This is of importance as the option of having a sterile product enables the start of the translation of this biofunctional coating towards preclinical and subsequently first-in-man applications. Acknowledgments: We acknowledge the financial support of the Federal Ministry of Education and Research, BMBF (13GW0452A-C)

The regenerative capacity of hyaline cartilage is greatly limited. To prevent the onset of osteoarthritis, cartilage defects have to be properly treated. Cartilage, tissue engineered by mean of bioactive glass (BG) scaffolds presents a promising approach. Until now, conventional BGs have been used mostly for bone regeneration, as they are able to form a hydroxyapatite (HA) layer and are therefore, less suited for cartilage reconstruction. The aim of this study is to compare two BGs based on a novel BG composition tailored specifically for cartilage (CAR12N) and patented by us with conventional BG (BG1393) with a similar topology. The highly porous scaffolds consisting of 100% BG (CAR12N, CAR12N with low Ca2+/Mg2+ and BG1393) were characterized and dynamically seeded with primary porcine articular chondrocytes (pACs) or primary human mesenchymal stem cells (hMSCs) for up to 21 days. Subsequently, cell viability, DNA and glycosaminoglycan contents, cartilage-specific gene and protein expression were evaluated. The manufacturing process led to a comparable high (over 80%) porosity in all scaffold variants. Ion release and pH profiles confirmed bioactivity for them. After both, 7 and 21 days, more than 60% of the total surfaces of all three glass scaffold variants was densely colonized by cells with a vitality rate of more than 80%. The GAG content was significantly higher in BG1393 colonized with pACs. In general, the GAG content was higher in pAC colonized scaffolds in comparison to those seeded with hMSCs. The gene expression of cartilage-specific collagen type II, aggrecan, SOX9 and FOXO1 could be detected in all scaffold variants, irrespectively whether seeded with pACs or hMSCs. Cartilage-specific ECM components could also be detected at the protein level. In conclusion, all three BGs allow the maintenance of the chondrogenic phenotype or chondrogenic differentiation of hMSCs and thus, they present a high potential for cartilage regeneration

Bone homeostasis is a highly regulated process involving pathways in bone as WNT, FGF or BMP, but also requiring support from surrounding tissues as vessels and nerves. In bone diseases, the bone-vessel-nerve triad is impacted. Recently, new players appeared as regulators of bone homeostasis: microRNAs (miRNA). Five miRNAs associated with osteoporotic fractures are already known, among which miR-125b is decreasing bone formation by downregulating human mesenchymal stem cells (hMSCs) differentiation. Other miRNAs, as miR-214 (in cluster with miR-199a), are secreted by osteoclasts to regulate osteoblasts and inhibit bone formation. This forms a very complex regulatory network. hMSCs and osteoblasts (n=3) were transfected with mimic/antagomiR of miR-125b, miR-199a-5p or miR-214, or with a scrambled miRNA (negative control) in osteogenic differentiation calcium-enriched medium (Ca++). Mineralization was assessed by Alizarin Red/CPC staining, miRNA expression by qPCR and protein by western blotting. Exposure of hMSCs or osteoblasts to Ca++ increased mineralization compared to basal medium. hMSCs transfected with miR-125b mimic in Ca++ presented less mineralization compared to scramble. This correlated with decreased levels of BMPR2 and RUNX2. hMSCs transfected with miR-125b inhibitor presented higher mineralization. Interestingly, hMSCs transfected with miR-214 mimic in Ca++ presented no mineralization while miR-214 inhibitor increased mineralization. No differences were observed in hMSCs transfected with miR-199a-5p modulators. On the contrary, osteoblasts transfected with miR-199a-5p mimic present less mineralization than scrambled-transfected and same was observed for miR-214 and miR-125b mimics. We highlight that miR-125b and miR-214 decrease mineralization of hMSCs in calcium-enriched medium. We noticed that miR-199a-5p is able to regulate mineralization in osteoblasts but not in hMSCs suggesting that this effect is cell-specific. Interestingly, the cluster miR-199a/214 is known as modulator of vascular function and could thus contribute to bone remodeling via different ways. With this work we slightly open the door to possible therapeutic approaches for bone diseases

Aims. Astragalus polysaccharide (APS) participates in various processes, such as the enhancement of immunity and inhibition of tumours. APS can affect osteoporosis (OP) by regulating the osteogenic differentiation of human bone mesenchymal stem cells (hBMSCs). This study was designed to elucidate the mechanism of APS in hBMSC proliferation and osteoblast differentiation. Methods. Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting were performed to determine the expression of microRNA (miR)-760 and ankyrin repeat and FYVE domain containing 1 (ANKFY1) in OP tissues and hBMSCs. Cell viability was measured using the Cell Counting Kit-8 assay. The expression of cyclin D1 and osteogenic marker genes (osteocalcin (OCN), alkaline phosphatase (ALP), and runt-related transcription factor 2 (RUNX2)) was evaluated using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Mineral deposits were detected through Alizarin Red S staining. In addition, Western blotting was performed to detect the ANKFY1 protein levels following the regulation of miR-760. The relationship between miR-760 and ANKFY1 was determined using a luciferase reporter assay. Results. The expression of miR-760 was upregulated in OP tissues, whereas ANKFY1 expression was downregulated. APS stimulated the differentiation and proliferation of hBMSCs by: increasing their viability; upregulating the expression levels of cyclin D1, ALP, OCN, and RUNX2; and inducing osteoblast mineralization. Moreover, APS downregulated the expression of miR-760. Overexpression of miR-760 was found to inhibit the promotive effect of APS on hBMSC differentiation and proliferation, while knockdown of miR-760 had the opposite effect. ANKFY1 was found to be the direct target of miR-760. Additionally, ANKFY1 participated in the APS-mediated regulation of miR-760 function in hBMSCs. Conclusion. APS promotes the osteogenic differentiation and proliferation of hBMSCs. Moreover, APS alleviates the effects of OP by downregulating miR-760 and upregulating ANKFY1 expression. Cite this article: Bone Joint Res 2023;12(8):476–485

Objectives. As one of the heat-stable enterotoxins, Staphylococcal enterotoxin C2 (SEC2) is synthesized by Staphylococcus aureus, which has been proved to inhibit the growth of tumour cells, and is used as an antitumour agent in cancer immunotherapy. Although SEC2 has been reported to promote osteogenic differentiation of human mesenchymal stem cells (MSCs), the in vivo function of SCE2 in animal model remains elusive. The aim of this study was to further elucidate the in vivo effect of SCE2 on fracture healing. Materials and Methods. Rat MSCs were used to test the effects of SEC2 on their proliferation and osteogenic differentiation potentials. A rat femoral fracture model was used to examine the effect of local administration of SEC2 on fracture healing using radiographic analyses, micro-CT analyses, biomechanical testing, and histological analyses. Results. While SEC2 was found to have no effect on rat MSCs proliferation, it promoted the osteoblast differentiation of rat MSCs. In the rat femoral fracture model, the local administration of SEC2 accelerated fracture healing by increasing fracture callus volumes, bone volume over total volume (BV/TV), and biomechanical recovery. The SEC2 treatment group has superior histological appearance compared with the control group. Conclusion. These data suggest that local administration of SEC2 may be a novel therapeutic approach to enhancing bone repair such as fracture healing. Cite this article: T. Wu, J. Zhang, B. Wang, Y. Sun, Y. Liu, G. Li. Staphylococcal enterotoxin C2 promotes osteogenesis of mesenchymal stem cells and accelerates fracture healing. Bone Joint Res 2018;7:179–186. DOI: 10.1302/2046-3758.72.BJR-2017-0229.R1

Minimally invasive surgery for the restoration of bone tissues lost due to diseases and trauma is preferred by the health care system as the related costs are continuously increasing. Recently, efforts have been paid to optimize injectable calcium phosphate (CaP) cements which have been recognized as excellent alloplastic material for osseous augmentation because of their unique combination of osteoconductivity, biocompatibility and mouldability. The sol-gel synthesis approach appears to be the most suitable route towards performing injectable calcium phosphates. Different strategies used to prepare bioactive and osteoinductive injectable CaP are reported. CaP gels complexed with phosphoserine-tethered poly(ε-lysine) dendrons (G3-K PS) designed to interact with the ceramic phase and able to induce osteogenic differentiation of human mesenchymal stem cells (hMSCs) is discussed. Recently, attention has been given to the modification of hydroxyapatite with Strontium (Sr) due to its dual mode of action, simultaneously increasing bone formation (stimulating osteoblast differentiation) while decreasing bone resorption (inhibiting osteoclast differentiation). The effect of systems based on strontium modified hydroxyapatite (Sr-HA) at different composition on proliferation and osteogenic differentiation of hMSC is described. One more approach is based on the use of antimicrobial injectable materials. It has been demonstrated that some imidazolium, pyridinium and quaternary ammonium ionic liquids (IL) have antimicrobial activity against some different clinically significant bacterial and fungal pathogens. Here, we report several systems based on IL at different alkyl-chain length incorporated in Hydroxyapatite (HA) through the sol-gel process to obtain an injectable material with simultaneous opposite responses toward osteoblasts and microbial proliferation

Purpose: Recent evidence indicates that a major drawback of current cartilage and intervertebral disc (IVD) tissue engineering is that human mesenchymal stem cells (MSCs) from osteoarthritic patients rapidly express type X collagen (COL10A1), a marker of late-stage chondrocyte hypertrophy associated with endochondral ossification. We recently discovered that a novel atmospheric-pressure plasma-polymerized thin film substrate, named “nitrogen-rich plasma-polymerized ethylene” (PPE:N), is able to inhibit COL10A1 expression in committed MSCs. However, the cellular mechanisms implicated in the inhibition of COL10A1 expression by PPE:N surfaces are unknown. Method: Human mesenchymal stem cells (MSCs) were obtained from aspirates from the intramedullary canal of donors (60–80 years of age) undergoing total hip replacement for osteoarthritis. Bone marrow aspirates were processed and MSCs were cultured on commercial polystyrene (PS control) and on PPE:N surfaces in the presence of different kinases and cyclooxygenase inhibitors for 3 days. Total RNA was extracted with TRIzol reagent (Invitrogen, Burlington, ON) and the expression of COL10A1, cyclooxygenase-1 (COX-1), and 5-lipoxygenase (5-LOX) genes was measured by real-time quantitative RT-PCR. Results: Results showed that a non-specific inhibitor of cyclooxygenases reduced the expression of COL10A1. In contrast, inhibitors of protein kinases stimulated the expression of COL10A1. Furthermore, potent and selective inhibitors of COX-1 and 5-LOX also reduced the expression of COL10A1. However, COX-2 and 12-LOX inhibitors had no significant effect on the expression of COL10A1. COX-1 gene expression was also decreased when MSCs were incubated on “S5” PPE:N surfaces. Interestingly, MSCs did not express 5-LOX. Conclusion: PPE:N surfaces suppress COL10A1 expression through the inhibition of COX-1 which is directly implicated in the synthesis of prostaglandins. The decreased expression of COX-1 and COL10A1 in human MSCs cultured on PPE:N is therefore in agreement with the induction of the osteogenic capacity of rat bone marrow and bone formation by systemic or local injection of PGE2 in rats. However, PGE2 and other prostaglandins inhibited COL10A1 expression in chick growth plate chondrocytes. This suggests that the effect of prostaglandins on COL10A1 expression may be cell-specific or may be dependent on pre-existing patho-physiological conditions

Metallic implants are widely used in orthopedic, oral and maxillofacial surgery. Durable osseous fixation of an implant requires that osteoprogenitor cells attach and adhere to the implant, proliferate, differentiate into osteoblasts, and produce mineralized matrix. We previously observed that human mesenchymal stem cells (MSCs) adherent to smooth tantalum (Ta) surfaces demonstrated superior biocompatibility compared with titanium (Ti) coatings. The aim of the present study was to investigate the interactions between MSCs and smooth surfaces of Ta and by means of whole-genome microarray technology. Immortalized human mesenchymal stem cells were cultivated on smooth surfaces of Ti and Ta. Total RNA was extracted after culturing for 1, 2, 4, and 8 days and hybridized to Affymetrix whole-genome microarrays (N=16). Replicate arrays were averaged and the ratios of gene expression by MSCs cultivated on Ta versus Ti coating were calculated. Absolute fold differences were also calculated and lists of upregulated genes were generated. Moreover, gene Ontology (GO) annotation analysis of differentially regulated genes was performed. For both Ta and Ti coatings, the vast majority of genes were upregulated after 4 d of cultivation. Genes upregulated by MSCs cultivated on Ta coating for 4 d were annotated to relevant GO terms. Ti-regulated GO annotation clusters were predominantly transcription-related. By using the K-means clustering algorithm, 10 clusters containing more than 5 genes were identified. Moreover, various genes related to osteogenesis and cell adhesion were upregulated by MSCs exposed to Ta surface. Microarray analysis of MSCs exposed to smooth metallic surfaces of both Ta and Ti generally showed a huge increase in transcriptional activity after 4 d of cultivation. According to GO annotation analysis Ta coating may induce increased adhesion and earlier differentiation of MSCs compared to Ti surface making Ta a promising biocompatible material for bone implants

Summary. Within hours after exposure to hypoxic circumstances hMSCs start producing AGFs. Initially hypoxia does not affect hMSC proliferation and metabolic activity, but after 7 days both are decreased, compared to hMSCs cultured under ambient oxygen conditions. Introduction. At the moment of implantation of a large cell seeded scaffold, usually a vascular network is lacking within the scaffold. Therefore, the cells seeded on the scaffold are exposed to hypoxic circumstances. Human mesenchymal stem cells (hMSCs) exposed to hypoxic circumstances, start to produce angiogenic factors (AGF). 1. and to proliferate faster than at ambient oxygen levels. 2. Under severe, continued hypoxia, hMSC metabolism slows down and ultimately stops. 3. We hypothesise that there is a threshold oxygen level above which hMSCs at hypoxia will both produce AGF and still proliferate, and below which cells slow down their metabolism. If hMSCs are provided with oxygen levels just above this threshold, effective tissue regeneration, which requires cell proliferation and vascular ingrowth, may be accomplished. Methods. Human mesenchymal stem cells (hMSCs) were isolated using Ficoll density gradient centrifugation from reaming debris that was collected during total hip replacement. Experiments were performed in a hypoxic chamber at 5% CO. 2. and 1%, 2%, 3%, 4%, 6%, 8% and 10% O. 2. , temperature was 37°C and humidity was 100%. The cells were transfected with a pCDNA\HRE-luciferase 2 plasmid to find the oxygen range at which hMSCs start producing AGF. Subsequently, AGF production was quantified using qPCR. Cell proliferation and cell metabolism rate under hypoxic circumstances was assayed using the CyQuant DNA quantification method and a (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, respectively. Results. In hMSCs cultured at 1% O. 2. , the hypoxia responsive element was activated. A selection of AGFs was expressed at 1%, 2% and 3% O. 2. AGF production started four hours after exposure to hypoxic circumstances commenced. Initially, at 1 day of exposure to hypoxia, cell proliferation and cell metabolic activity were not influenced by hypoxia. After 7 days, hMSC proliferation and metabolic activity were lower in cells grown under hypoxic circumstances than in cells grown under ambient oxygen circumstances. Discussion/Conclusion. The threshold level above which hMSCs at hypoxia will both produce AGF and still proliferate seems lower than 1% O. 2. Within hours after exposure to hypoxic circumstances hMSCs start producing AGFs. Initially, hMSC proliferation and metabolic activity were not affected by hypoxia, but after 7 days both proliferation and metabolic activity were decreased compared to hMSCs cultured under ambient oxygen circumstances

Objectives. Regenerative medicine is an emerging field aimed at the repair and regeneration of various tissues. To this end, cytokines (CKs), growth factors (GFs), and stem/progenitor cells have been applied in this field. However, obtaining and preparing these candidates requires invasive, costly, and time-consuming procedures. We hypothesised that skeletal muscle could be a favorable candidate tissue for the concept of a point-of-care approach. The purpose of this study was to characterize and confirm the biological potential of skeletal muscle supernatant for use in regenerative medicine. Methods. Semitendinosus muscle was used after harvesting tendon from patients who underwent anterior cruciate ligament reconstructions. A total of 500 milligrams of stripped muscle was minced and mixed with 1 mL of saline. The collected supernatant was analysed by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. The biological effects of the supernatant on cell proliferation, osteogenesis, and angiogenesis in vitro were evaluated using human mesenchymal stem cells (hMSCs) and human umbilical cord vein endothelial cells (HUVECs). Results. The supernatant contained several GFs/CKs, with especially high levels of basic fibroblast growth factor, and CD34+ cells as the stem/progenitor cell fraction. With regard to biological potential, we confirmed that cell proliferation, osteoinduction, and angiogenesis in hMSCs and HUVECs were enhanced by the supernatant. Conclusions. The current study demonstrates the potential of a new point-of-care strategy for regenerative medicine using skeletal muscle supernatant. This attractive approach and readily-available material could be a promising option for tissue repair/regeneration in the clinical setting. Cite this article: M. Yoshikawa, T. Nakasa, M. Ishikawa, N. Adachi, M. Ochi. Evaluation of autologous skeletal muscle-derived factors for regenerative medicine applications. Bone Joint Res 2017;6:277–283. DOI: 10.1302/2046-3758.65.BJR-2016-0187.R1

The demographic challenges of an advancing aged population emphasise the need for innovative approaches to tissue reconstruction to augment and repair tissue lost as a consequence of trauma or degeneration. Currently, the demand for bone graft outstrips supply, a key issue in the field of revision hip surgery where impaction bone grafting of the femur and acetabulum has impressive results in the short and medium term but often requires up to 6 donated femoral heads. Spine and selected tumour and trauma cases are also eminently suitable for this mode of bone stock replacement. In the current study, we examined the histological and biochemical findings of two parallel in-vitro and in-vivo studies using human mesenchymal stem cells on synthetic scaffolds for possible bone augmentation. The first study confirmed that culture expanded bone marrow cells from 3 patients (mean age 76 +/−4) could be successfully seeded onto washed morsellised allograft. The seeded graft was then exposed to a force equivalent to a standard femoral impaction (impulse=474 J/m2) and cultured for 4 weeks in osteogenic media. Examination of cell viability using cell tracker green and ethidium homodimer-1 and confocal microscopy confirmed extensive cell proliferation and viability following impaction and culture. Alcian blue/ Sirius red confirmed matrix production, alkaline phosphatase immunocytochemistry production of enzyme activity and Goldners trichrome enhanced osteoid formation. The second study compared 3 scaffolds; bone allograft, a ß – Tricalcium Phosphate (ß-TCP) graft substitute and a 50:50 mixture of allograft and ß-TCP. The scaffolds were seeded with either immunoselected STRO-1+ human mesenchymal stem cells or unselected marrow cells. The scaffolds were similarly exposed to impaction forces and cultured for 4 weeks in vitro or in vivo, implanted subcutaneously in MF1nu/nu mice. Both studies demonstrated cellular viability, activity and osteogenesis as assessed using confocal microscopy, Goldners trichrome and alcian blue/Sirius cytochemistry. The demonstration of enhanced osteoid formation as a consequence of stem cell proliferation after impaction grafting augers well for the success of autologous stem cell implantation on impacted graft substitute with or without the addition of morsellised allograft. The implications therein for clinical use in the future await clinical trials

Introduction: The use of uncemented arthroplasty in joint replacement surgery requires osseointegration of the prosthesis to maximise function and longevity. It has been demonstrated that osteoblast-like cells will preferentially proliferate, differentiate and produce mineralised matrix in pits and grooves on non-biological surfaces, of similar dimensions to those of Howslip’s lacunae produced by osteoclasts in vitro. The hypotheses of this study were that a photochemically etched titanium alloy surface would 1) induce proliferation and differentiation in osteoblast-like cells; 2) induce osteoblastic differentiation of human mesenchymal stem cells and 3) induce greater bone to implant contact in a caprine model. Methods: Three microgrooved titanium alloy surfaces (fine, medium & coarse) were created by photochemical etching, with dimensions of 200 to 515 microns. Human Mesenchymal stem cells (MSC) and Human Osteosarcoma (HOS) cells (TE-85) were seeded onto these surfaces and cultured in standard media; in the case of MSC, with and without the addition of osteogenic supplements. At intervals of time each surface and cell type were assessed for proliferation by Alamar blue assay and osteoblastic differentiation by Alkaline Phosphatase expression. A polished titanium surface was used as a control. A plate of each surface dimension was placed into a femoral condyle of ten adult male goats. The animals were euthanased at 6 and 12 weeks post-implantation. The specimens were histologically processed and examined under light and backscattered electron microscopy to establish the percentage of bone to implant contact and the presence of new bone within the grooves. Results: In vitro, all cells showed an increase in proliferation with time, the greatest occurring on the coarse surface. Alkaline phosphatase expression showed a rise with time on all surfaces, the greatest being on the coarse surface seeded with HOS cells (p< 0.05). MSC could not be induced to differentiate to an osteogenic lineage by these surface textures alone. On addition of osteogenic supplements their results followed the trends of HOS cells. In vivo, histomorphometric analysis showed significantly greater bone implant contact on the coarse surface at both 6 and 12 weeks (p< 0.05). In a number of cases there were signs of osteogenesis occurring deep within the pits and grooves. Discussion: This study confirms that a photochemically etched surface topography mimicking that created by osteoclasts will increase the proliferation and differentiation of osteoblastic cells in vitro. The rate of differentiation of these cells increased significantly in relation to the size of the grooves. When implanted in vivo these same surfaces were shown to support osseointegration. This surface has the potential to improve the function of uncemented arthroplasties in the future

The use of stem cells transplanted into the intervertebral disc (IVD) is a promising regenerative approach to treat intervertebral disc degeneration (IDD). The aim of this study was to assess the effect of a hydrogel composed of hyaluronic acid (HA) and platelet-rich plasma (PRP) loaded with human mesenchymal stem cells (hMSCs), on IVD extracellular matrix synthesis and nucleus pulposus (NP) marker expression in a whole IVD culture model. HA was blended with batroxobin (BTX), a gelling agent activated in presence of PRP to construct a hydrogel. Bovine IVDs (n=25) were nucleotomised and filled with 1×10. 6. or 2×10. 6. hMSCs suspended in ∼150 mL of the PRP/HA/BTX hydrogel. IVDs harvested at day 0 and nucleotomised IVDs with no hMSCs and/or hydrogel were used as controls. hMSCs alone or encapsulated in the hydrogel were also cultured in well plates to examine the effect of the IVD microenvironment on hMSCs. After 1 week, tissue structure, scaffold integration and gene expression of anabolic (collagen type I, collagen type II and aggrecan), catabolic (matrix metalloproteinase 3 – MMP-3 –, MMP-13 and a disintegrin and metalloproteinase with thrombospondin motifs 4) and NP cell (cytokeratin 19, carbonic anhydrase 12, cluster of differentiation 24) markers were assessed. Histological analysis showed a good integration of the scaffold within the NP area with cell repopulation. At the gene expression level, the hMSC-loaded hydrogels demonstrated to increase disc cell anabolic and catabolic marker expression and promoted hMSC differentiation towards a NP cell phenotype. This study demonstrated that the HA/PRP/BTX may represent a valid carrier for hMSCs being capable of stimulating cell activity and NP marker expression as well as achieving a good integration with the surrounding tissues

Aims. The purpose of this study was to explore a simple and effective method of preparing human acellular amniotic membrane (HAAM) scaffolds, and explore the effect of HAAM scaffolds with juvenile cartilage fragments (JCFs) on osteochondral defects. Methods. HAAM scaffolds were constructed via trypsinization from fresh human amniotic membrane (HAM). The characteristics of the HAAM scaffolds were evaluated by haematoxylin and eosin (H&E) staining, picrosirius red staining, type II collagen immunostaining, Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM). Human amniotic mesenchymal stem cells (hAMSCs) were isolated, and stemness was verified by multilineage differentiation. Then, third-generation (P3) hAMSCs were seeded on the HAAM scaffolds, and phalloidin staining and SEM were used to detect the growth of hAMSCs on the HAAM scaffolds. Osteochondral defects (diameter: 3.5 mm; depth: 3 mm) were created in the right patellar grooves of 20 New Zealand White rabbits. The rabbits were randomly divided into four groups: the control group (n = 5), the HAAM scaffolds group (n = 5), the JCFs group (n = 5), and the HAAM + JCFs group (n = 5). Macroscopic and histological assessments of the regenerated tissue were evaluated to validate the treatment results at 12 weeks. Results. In vitro, the HAAM scaffolds had a network structure and possessed abundant collagen. The HAAM scaffolds had good cytocompatibility, and hAMSCs grew well on the HAAM scaffolds. In vivo, the macroscopic scores of the HAAM + JCFs group were significantly higher than those of the other groups. In addition, histological assessments demonstrated that large amounts of hyaline-like cartilage formed in the osteochondral defects in the HAAM + JCFs group. Integration with surrounding normal cartilage and regeneration of subchondral bone in the HAAM + JCFs group were better than those in the other groups. Conclusion. HAAM scaffolds combined with JCFs promote the regenerative repair of osteochondral defects. Cite this article: Bone Joint Res 2022;11(6):349–361

Introduction and Objective. Alveolar bone resorption following tooth extraction or periodontal disease compromises the bone volume required to ensure the stability of an implant. Guided bone regeneration (GBR) is one of the most attractive technique for restoring oral bone defects, where an occlusive membrane is positioned over the bone graft material, providing space maintenance required to seclude soft tissue infiltration and to promote bone regeneration. However, bone regeneration is in many cases impeded by a lack of an adequate tissue vascularization and/or by bacterial contamination. Using simultaneous spray coating of interacting species (SSCIS) process, a bone inspired coating made of calcium phosphate-chitosan-hyaluronic acid was built on one side of a nanofibrous GBR collagen membrane in order to improve its biological properties. Materials and Methods. First, the physicochemical characterizations of the resulting hybrid coating were performed by scanning electron microscopy, X-ray photoelectron, infrared spectroscopies and high-resolution transmission electron microscopy. Then human mesenchymal stem cells (MSCs) and human monocytes were cultured on those membranes. Biocompatibility and bioactivity of the hybrid coated membrane were respectively evaluated through MSCs proliferation (WST-1 and DNA quantification) and visualization; and cytokine release by MSCs and monocytes (ELISA and endothelial cells recruitment). Antibacterial properties of the hybrid coating were then tested against S. aureus and P. aeruginosa, and through MSCs/bacteria interactions. Finally, a preclinical in vivo study was conducted on rat calvaria bone defect. The newly formed bone was characterized 8 weeks post implantation through μCT reconstructions, histological characterizations (Masson's Trichrome and Von Kossa stain), immunohistochemistry analysis and second harmonic generation. Biomechanical features of newly formed bone were determined. Results. The resulting hybrid coating of about 1 μm in thickness is composed of amorphous calcium phosphate and carbonated poorly crystalline hydroxyapatite, wrapped within chitosan/hyaluronic acid polysaccharide complex. Hybrid coated membrane possesses excellent bioactivity and capability of inducing an overwhelmingly positive response of MSCs and monocytes in favor of bone regeneration. Furthermore, the antibacterial experiments showed that the hybrid coating provides contact-killing properties by disturbing the cell wall integrity of Gram-positive and Gram-negative bacteria. Its combination with MSCs, able to release antibacterial agents and mediators of the innate immune response, constitutes an excellent strategy for fighting bacteria. A preclinical in vivo study was therefore conducted in rat calvaria bone defect. μCT reconstructions showed that hybrid coated membrane favored bone regeneration, as we observed a two-fold increase in bone volume / total volume ratios vs. uncoated membrane. The histological characterizations revealed the presence of mineralized collagen (Masson's Trichrome and Von Kossa stain), and immunohistochemistry analysis highlighted a bone vascularization at 8 weeks post-implantation. However, second harmonic generation analysis showed that the newly formed collagen was not fully organized. Despite a significant increase in the elastic modulus of the newly formed bone with hybrid coated membrane (vs. uncoated membrane), the obtained values were lower than those for native bone (approximately 3 times less). Conclusions. These significant data shed light on the regenerative potential of such bioinspired hybrid coating, providing a suitable environment for bone regeneration and vascularization, as well as an ideal strategy to prevent bone implant-associated infections