Background: Traditionally, it is believed that structural failure of the ischemic epiphysis as well as changes in radiodensity seen in Legg-Calve-Perthes disease are due to repair. Little is known if bone material properties are altered following ischemic necrosis of the juvenile femoral head. Purpose of this study was to determine bone matrix mineralization density, an important determinant of bone quality and strength, in an experimental model of juvenile ischemic osteonecrosis. Methods: Ten piglets were surgically induced with ischemic osteonecrosis and euthanized at 4- and 8 weeks following surgery. Contralateral, unoperated femoral heads were used as controls. Bone Mineralization Density Distribution (BMDD) parameters were determined using quanitative backscattered electron imaging (qBEI) in the epiphyseal calcified cartilage, subchondral and central trabecular bone region. Histological assessment was also performed. Results: In necrotic calcified epiphyseal cartilage matrix as well as subchondral bone matrix, a significant increase in the degree (CaMean, Ca Peak) as well as the homogeneity of mineralization (CaWidth reduction) and a significantly reduced amount of low mineralized matrix (CaLow) were observed at 4 and 8 weeks post ischemia induction. In the necrotic central trabecular region a significant increase in the degree and homogeneity of mineralization, as well as a decrease in the amount of low mineralized bone was found at 8 weeks post-ischemia induction, but not at 4-weeks, indicating that changes in necrotic trabecular bone occur more slowly. Changes in the necrotic calcified cartilage region were more dramatic than in necrotic bone. Discussion: Our findings indicate that the mineralization process continues in the necrotic calcified cartilage and bone following femoral head infarction. This leads to an increased degree and homogeneity of mineralization in calcified cartilage and bone matrices and therefore altered material properties. These alterations in matrix mineralization status would lead to more brittle bone, prone to micro-fractures and may partly explain the weakening of structural properties of necrotic bone. Moreover, an increase in calcified cartilage and bone mineralization may also explain the increased radiodensity seen in the early stage of Perthes disease prior to repair and/or structural failure

Aims. Gene therapy research in the field of orthopaedics and traumatology have evolved during the last decade, leading to possible applications for the treatment of pathological conditions, such as bone fractures and cartilage defects. In particular, several gene transfer techniques have been employed so far for inducing bone formation in animal models of bone defects. Cell-based approaches, using in vitro and ex vivo genetically modified cells to be implanted in the animal, produced promising results as they enable the production of physiologic doses of an osteoinductive gene product into selected anatomical sites. In this study we used autologous skin fibroblasts, which are very simple to harvest and propagate in culture, transduced ex vivo with the new osteo-genic factor Lim Mineralization Factor-3 (Ad-LMP-3). These engineered cells produced successful bone healing when implanted by the use of a scaffold in rats, validating the in vivo osteoinductive properties of hLMP-3. Methods. Primary dermal fibroblasts cultures were established using a 1cm. 2. biopsy of shaved skin obtained from the abdomen of each rat after anesthesia. Semi-confluent primary fibroblasts were infected with either AdBMP-2 or AdhLMP-3 or both, using a overall multiplicity of infection (MOI) of 100 viral particles per cell. Cells transduced with Ad-eGFP at the same MOI were used as a viral infection control, while untreated cells served as a negative control. The transduced cells were harvested 24 hours after viral infection, resuspended in sterile PBS, let adsorbed on a Hydroxyapatite/Collagen scaffold and then implanted in a bone defect surgically performed in the mandible of immunocompetent rats. The animals were divided in 4 groups: 9 rats were treated with cells infected with AdLMP-3, 9 rats with cells infected with AdBMP-2 (positive controls), 9 rats with cells transduced with Ad-eGFP and 9 rats with untreated cells (controls). 3 Rats from each group were sacrified at 1, 2 and 3 months after the treatment and studied by x-rays, Micro-CT and histology (Von kossa and Alizarin staining). Results. All the animals treated with LMP-3 showed healing of the bone defect after 3 months, as confirmed his-tologically and radiographically. On the contrary none of the controls showed bone formation at latest time point. Conclusions. Recently, Lim Mineralization Proteins (LMP), coded by three different splice variants (LMP-1, LMP-2, LMP-3) of the same gene, have been identified as regulators of the osteoblast differentiation program. We have previously demonstrated that human LMP-3 (hLMP-3) contributes actively to bone formation, acting at least in part, through the BMP-2 signaling pathway, being capable of inducing differentiation of cells of mes-enchymal derivation towards the osteoblastic lineage, through the up-regulation of bone-specific genes, along with ectopic bone formation in vivo and mineralization in vitro. In this study we have analyzed the efficacy of an ex-vivo approach using autologous dermal fibroblasts infected with AdLMP-3. Engineered cells produced bone healing when implanted by the use of a scaffold in a rodent model, validating the in vivo osteoinductive properties of hLMP-3

Matrix-bound vesicles (MBVs) are embedded within osteoid and function as the site of initial mineral formation. However, they remain insufficiently characterised in terms of biogenesis, composition and function while their relationship with secreted culture medium EVs (sEVs) such as exosomes remains debated. We aimed to define the biogenesis and pro-mineralisation capacity of MBVs and sEVs to understand their potential in regenerative orthopaedics.

sEVs and MBVs isolated from conditioned medium (differential ultracentrifugation) and ECM (collagenase digestion and differential ultracentrifugation) of mineralising MC3T3 pre-osteoblast and human bone marrow MSC cultures were characterised by nanoparticle tracking analysis, western blotting, nano-flow cytometry, super resolution microscopy (ONI) and TEM. Immunoprecipitated populations positive for alkaline phosphatase (ALP), a putative marker of mineralisation capacity, were also characterised. Collagen binding efficiency was evaluated using MemGlow staining.

Results reported were comparative across both cell lines. Western blots indicated MBV fractions were positive for markers of endosomal biogenesis (CD9, CD81, ALIX, TSG101) and pro-mineralising proteins (ALP, Pit1, Annexin II, Annexin V), with Annexin V and CD9 present in immunoprecipitated ALP-positive fractions. MBVs were significantly larger than sEVs (p<0.05) and contained a higher amount of ALP (p<0.05) with a significant increase from day 7 to day 14 of cellular mineralisation (p<0.05). This mirrored the pattern of electron-dense vesicles seen via TEM. Super resolution single vesicle analysis revealed for the first-time co-expression of ALP with markers of endosomal biogenesis (CD9, CD63, CD81, ALIX) and Annexin II in both vesicle types, with higher co-expression percentage in MBVs than sEVs. MBVs also exhibited preferential collagen binding.

Advanced imaging methods demonstrated that contrary to opinions in the field, MBVs appear to possess exosomal markers and may arise via endosomal biogenesis. However, it was evident that a higher proportion of MBVs possessed machinery to induce mineralisation and were enriched in mineral-dense material.

The implantation of endoprosthesis is a routine procedure in orthopaedics. Endoprosthesis are mainly manufactured from ceramics, polymers, metals or metal alloys. To ensure longevity of the implants they should be as biocompatible as possible and ideally have antibacterial properties, to avoid periprosthetic joint infections (PJI). Various antibacterial implant materials have been proposed, but have so far only been used sporadically in patients. PJI is one of the main risk factors for revision surgeries. The aim of the study was to identify novel implant coatings that both exhibit antibacterial properties whilst having optimal biocompatibility.

Six different novel implant coatings and surface modifications (EBM TiAl6V4, strontium, TiCuN, TiNbN, gentamicin phosphate (GP), gentamicin phosphate+cationic polymer (GP+CP)) were compared to standard CoCrMo-alloy. The coatings were further characterized with regard to the surface roughness. E. coli and S. capitis were cultured on the modified surfaces to investigate the antibacterial properties. To quantify bacterial proliferation the optical density (OD) was measured and viability was determined using colony forming units (CFU). Murine bone marrow derived macrophages (BMMs) were cultured on the surfaces and differentiated into osteoblasts to quantify the mineralisation using the alizarin red assay.

All novel coatings showed reduced bacterial proliferation and viability compared to standard CoCrMo-alloy. A significant reduction was observed for GP and GP+CP coated samples compared to CoCrMo (OD_GP,E.coli = 0.18±0.4; OD_GP+CP,E.coli = 0.13±0.3; p≤0.0002; N≥7-8). An increase in osteoblast-mediated mineralisation was observed on all surfaces tested compared to CoCrMo. Furthermore, GP and GP+CP coated samples showed a statistically significant increase (M_GP = 0.21±0.1; M_GP+CP = 0.25±0.2; p<0.0001; N≥3-6).

The preliminary data indicates that the gentamicin containing surfaces have the most effective antibacterial property and the highest osseointegrative capacity. The use of antibiotic coatings on prostheses could reduce the risk of PJI while being applied on osseointegrative implant surfaces.

Aim

Implant-associated infection usually require prolonged treatment or even removal of the implant. Local application of antibiotics is used commonly in orthopaedic and trauma surgery, as it allows reaching higher concentration in the affected compartment, while at the same time reducing systematic side effects. Ceftriaxone release from calcium sulphate has a particularly interesting, near-constant release profile in vitro, making it an interesting drug for clinical application. Purpose of the present study was to investigate the potential cytotoxicity of different ceftriaxone concentrations and their influence on osteogenic differentiation of human pre-osteoblasts.

Engineered cartilage is poorly organized and fails to recapitulate physiologic organization in a hyaline upper and a mineralizing bottom zone deemed important for proper function. Objective was to grow bizonal human cartilage constructs in which in vivo mineralization is self-restricted to the bottom zone. Self-assembling biomaterial-free cell discs were generated from mesenchymal stroma cells and allowed to accumulate proteoglycans and collagen-type II over 3 weeks. In vitro mineralization of the cell discs with four mineralization media for up to 8 weeks showed that calcification was supported in all media containing ß-glycerophosphate. However, proteoglycans were retained only in media containing insulin. Bizonal cartilage constructs were made from 3-week non-mineralized cell discs overlaid with chondrocyte-seeded starPEG-heparin hydrogel or with a fibrin-gel layer to select the best design for upper zone development. Freshly prepared zonal constructs were implanted into subcutaneous pouches of immuno-deficient mice to compare in vivo development. After 6 weeks in vivo, both construct types were rich in collagen-type II in the upper zone and contained a mineralized bottom zone. However, solely for starPEG constructs, tissue volume of the upper zone remained high and alkaline phosphatase, alizarin red, and collagen-type X staining were restricted to the bottom zone. StarPEG zonal constructs were superior to fibrin constructs due to self-restriction of mineralization and hypertrophic markers to the bottom zone. This innovative design of bizonal constructs offers the successful generation of an organized cartilage resembling the native cartilage with the chance for immediate use of autogenous chondrocytes in a one-step surgical joint intervention.

Background

Heterotopic ossification (HO) is lamellar bone formation in the soft tissues following trauma or joint replacement for osteoarthritis (OA). A genome wide association study of HO patients after total hip arthroplasty for OA has identified Kinesin Family Member 26B (KIF26B) as a gene associated with HO severity. KIF26B has previously been associated with HO in mice.

Hypothesis and aims: We hypothesised that Kif26b regulates the osteogenic trans-differentiation of myoblasts; a possible mechanism of HO. Using an in vitro model, we wished to establish whether Kif26b is involved in HO formation and to explore the molecular mechanism.

Osteophyte deposition and subchondral bone damage are notable features of osteoarthritis (OA). Deregulated mineralization contributes to osteophyte and subchondral irregularity. The microRNA-29 (miR-29) family is associated with arthritic disorders. This study is aimed to investigate miR-29a function to OA osteophyte formation and subchondral integrity. Intact and damaged articular cartilage in patients with end-stage knee OA who required total knee arthroplasty were harvested to probe miR-29a, cartilage, and mineralized matrix expression using RT-PCR and in situ hybridization. Osteophyte volume and subchondral morphometry of collagenase-induced OA knees in mice were quantified using μCT and histomorphometry. Increased bone matrix expression (collagen I and bone alkaline phosphatase) and reduced cartilage matrix (collagen II and aggrecan) along with low miR-29a expression existed in human OA specimens. Aged miR-29a knockout mice showed spontaneous osteophyte formation and articular cartilage erosion. In primary articular chondrocytes, miR-29a deficiency significantly reduced cartilage matrix synthesis, whereas von Kossa staining-positive mineralized matrix production was increased. Of interest, the severity of collagenase-induced osteophyte accumulation and subchondral damage along with serum cartilage breakdown products CTX-II and COMP levels were significantly compromised in mice overexpressing miR-29a. Intra-articularly injecting miR-29a significantly reduced osteophyte volume and subchondral integrity and retained cartilage morphology in collagenase-injured knees. Reduced miR-29a signalling worsens osteophyte and subchondral destruction in OA through increasing mineralized matrix formation of chondrocytes. Restoring miR-29a shields joints from cartilage degradation, osteophyte and subchondral destruction. This study conveys new mechanistic underlying OA osteophyte pathogenesis and shines light on the remedial potential of miR-29a to OA.

During remodelling, osteoclasts produce discrete bone cavities filled with bone and this is associated with the dimensions of the cavity. The aim of this study is to investigate the effect of pores of similar size to those produced by osteoclasts on the morphology, proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in vitro. The hypothesis is that a porous surface similar in morphology to a bone surface prepared by osteoclasts will increase cell proliferation and osteogenic differentiation of MSCs.

Sheep BMSCs were seeded onto plain titanium surfaces and 100µm, 250µm and 500µm discrete pores surfaces. Cell metabolic activity was investigated using Presto Blue on days 3, 7 and 10. Bone mineralisation was quantified by Alizarin red staining at days 3, 7 and 14. Cell morphology was observed by scanning electron microscopy (SEM). Data was statistically analysed using one-way analysis of variance and a Bonferroni correction method.

Cells on porous discs had a three dimensional phenotype and aligned on the circumference of each pore. Metabolic activity was significantly higher by day 10 on plain discs compared to all porous discs. Bone mineralization was significantly higher on 100µm pores by day 3 (0.545mM±0.66; p=0.047) than plain discs and significantly higher on both 100µm and 250µm pores by day 7(p=0.000 and p=0.005) than plain discs. Substantial mineralised bone matrix was found on 100µm discs without being treated with osteogenic supplements, compared to other control disc types (p=0.043, p=0.003, p=0.000).

The different topographies altered cell behaviour and migration.100µm pores demonstrated earlier and enhanced bone mineralisation even in the absence of osteogenic supplements. This pore size is aligned to the size of individual resorption bays that osteoclasts produce on bone surfaces and is considerably lower than the pore sizes used to enhance osteo-integration of implant surfaces.

Aims

Bone regeneration during treatment of staphylococcal bone infection is challenging due to the ability of Staphylococcus aureus to invade and persist within osteoblasts. Here, we sought to determine whether the metabolic and extracellular organic matrix formation and mineralization ability of S. aureus-infected human osteoblasts can be restored after rifampicin (RMP) therapy.

Purpose: After implantation of a total knee arthroplasty (TKA) for osteoarthritis, early demineralisation of the superior tibial epiphysis occurs with modification of bone mineral density (BMD) in the two compartments. The long-term trend is not known to date. We report the results of 38 prostheses followed prospectively with densitometric measurements at minimum five years.

Material and methods: This prospective analysis included 38 TKA implanted for primary degenerative disease in patients with a mean age of 70±4 years at implantation, 60% women. Clinical assessment (IKS) and radiography (HKA) as well as osteodensitometry were recorded before surgery, at six months, one year, two years and five years. Bone mineral status was assessed using the densitometry of the femoral neck before surgery and at five years for all patients. The DEXA method was used for each knee on the anteroposterior film. Seven zones were defined around the tibial implant, in particular two under the medial and lateral plateaus, and under the stem. We studied changes in each zone over time. The alpha risk was set at 5%. Inter- and intraobserver reproducibility was 2.9% and 2.8% respectively.

Results: At five years follow-up, the mean IKS function score was 85±19 and the knee score was 918. The mean mechanical axis was 180±2° with symmetrical distribution. There were no progressive lucent lines. The BMD of the homolateral femoral neck did not change significantly (0.763 g/cm2 at inclusion and 0.750 g/cm2 at five years), unlike the natural evolution in a reference population (women -4.5%, men -2.4%). The mean BMD in the seven zones decreased significantly (11.6%, p< 0.0001. Mean BMD was 0.936 g/cm2 at inclusion and 0.863 g/cm2 at six months), 0.823 g/cm2 at five years. BMD decreased very strongly from 0 to 6 months (−6.51%, p< 0.0001) then more slowly to the end of the first year (−3%) and finally declining regularly, but non-significantly, at a slower rate from 1 to 5 years. Study of the seven zones showed a difference in changes in the BMD between the medial, lateral and stem zones. The two medial zones decreased significantly from 6.33% to 6.18% especially during the first year (−2.06% and −2.09%) and more moderately from 1 to 5 years (−1.6%, −2.65%). The lateral zones showed a greater average decline in BMD (−10.5%, −8.92%) between 0 and 5 years: −8.57% and −6.75% during the first year then at a slower rate. The greatest loss in BMD was found under the stem; −14.3% at five years. Here again, between 0 and 6 months the decline was rapid: −8.09%. It reached −12.74% at one year then varied little, −1% and −2% between 1 and 5 years.

Conclusion: 1) Bone remodelling under the tibial base plate occurs early after implantation of a TKA. It occurs during the first year (especially the first six months). 2) Remodelling is more pronounced laterally than medially (good realignment but persistent varus stress with greater stress on the medial than lateral side). 3) The greatest loss in BMD occurs under the stem. 4) BMD of the femoral neck remains stable, unlike the evolution observed in a reference population.

Despite its intrinsic ability to regenerate form and function after injury, bone tissue can be challenged by a multitude of pathological conditions. While innovative approaches have helped to unravel the cascades of bone healing, this knowledge has so far not improved the clinical outcomes of bone defect treatment. Recent findings have allowed us to gain in-depth knowledge about the physiological conditions and biological principles of bone regeneration. Now it is time to transfer the lessons learned from bone healing to the challenging scenarios in defects and employ innovative technologies to enable biomaterial-based strategies for bone defect healing. This review aims to provide an overview on endogenous cascades of bone material formation and how these are transferred to new perspectives in biomaterial-driven approaches in bone regeneration.

Cite this article: T. Winkler, F. A. Sass, G. N. Duda, K. Schmidt-Bleek. A review of biomaterials in bone defect healing, remaining shortcomings and future opportunities for bone tissue engineering: The unsolved challenge. Bone Joint Res 2018;7:232–243. DOI: 10.1302/2046-3758.73.BJR-2017-0270.R1.

Aims. Continuous local antibiotic perfusion (CLAP) has recently attracted attention as a new drug delivery system for orthopaedic infections. CLAP is a direct continuous infusion of high-concentration gentamicin (1,200 μg/ml) into the bone marrow. As it is a new system, its influence on the bone marrow is unknown. This study aimed to examine the effects of high-concentration antibiotics on human bone tissue-derived cells. Methods. Cells were isolated from the bone tissue grafts collected from six patients using the Reamer-Irrigator-Aspirator system, and exposed to different gentamicin concentrations. Live cells rate, apoptosis rate, alkaline phosphatase (ALP) activity, expression of osteoblast-related genes, mineralization potential, and restoration of cell viability and ALP activity were examined by in vitro studies. Results. The live cells rate (the ratio of total number of cells in the well plate to the absorbance-measured number of live cells) was significantly decreased at ≥ 500 μg/ml of gentamicin on day 14; apoptosis rate was significantly increased at ≥ 750 μg/ml, and ALP activity was significantly decreased at ≥ 750 μg/ml. Real-time reverse transcription-polymerase chain reaction results showed no significant decrease in the ALP and activating transcription factor 4 transcript levels at ≥ 1,000 μg/ml on day 7. Mineralization potential was significantly decreased at all concentrations. Restoration of cell viability was significantly decreased at 750 and 1,000 μg/ml on day 21 and at 500 μg/ml on day 28, and ALP activity was significantly decreased at 500 μg/ml on day 28. Conclusion. Our findings suggest that the exposure concentration and duration of antibiotic administration during CLAP could affect cell functions. However, further in vivo studies are needed to determine the optimal dose in a clinical setting. Cite this article: Bone Joint Res 2024;13(3):91–100

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

Aim: To describe the radiographic findings of soft tissue sarcoma. Materials and Method: The retrospective review of 100 consecutive patients with a histological diagnosis of primary soft tissues sarcoma of the extremities. Results: Fifty five patients had plain radiographs at initial presentation. This was mainly due to the fact that most patients were tertiary referrals or had other initial imaging. Histological diagnosis in these patients was: liposarcoma in 24 patients, leiomyosarcoma in 8, undifferentiated spindle cell sarcoma in 5, malignant schwannoma in 4, synovial sarcoma in 4, MFH in 2, fibrosarcoma in 2, haemangiopericytoma, epithelioid sarcoma, malignant GCT, melanoma and spindle cell histiocytoma in one. The upper limb was involved in 18 patients and the lower limb was involved in 37. Thirty-five (63.6%) patients had a visible soft tissue mass on plain film. Eleven had mineralisation within the soft tissue mass and seven had either bone involvement or periosteal response. Those with a distinct soft tissue mass and evidence of fat content on plain film were noted to be diagnosis of liposarcoma in 86.7% of the cases. Mineralization was noted in synovial sarcoma (2), liposarcoma (3), leiomyosarcoma (1), MFH (2) and poorly differentiated sarcomas (2). Conclusion: The plain radiograph is useful in assessing soft tissue tumour and abnormality is seen in 2/3 of cases reviewed. Mineralization as a radiographic finding features in malignant sarcoma notably liposarcoma. With tumours demonstrating fat on plain film this can correlate with MRI and facilitate surgical treatment avoiding biopsy

Chondrogenic differentiation and cartilage homeostasis requires a high cellular translational capacity to meet the demands for cartilaginous extracellular matrix production. Box C/D and H/ACA snoRNAs guide post-transcriptional 2′-O ribose methylation and pseudouridylation of specific ribosomal RNA (rRNA) nucleotides, respectively. How specific rRNA modifications influence rRNA function is poorly documented, but modifications are thought to tune rRNA folding and interaction with ribosomal proteins, which is critical for ribosome function. We hypothesise that chondrocyte translational capacity is supported by snoRNA-mediated post-transcriptional fine-tuning of rRNAs. ATDC5 progenitor cells were differentiated into the chondrogenic lineage, resembling mature and mineralising chondrocytes after 7 or 14 days, respectively. UBF-1 (rRNA transcription factor), fibrillarin (box C/D methyltransferase) and dyskerin (box H/ACA pseudouridylase) expression displayed highest fold induction at day 5/6 in differentiation. Ribosomal RNA content per cell was increased at day 7, but not at day 14 in differentiation. These data suggest that ribosome biogenesis adapts to the chondrocyte's differentiation status. RNA-Seq of RNA species <200 nt revealed expression of at least 224 individual snoRNAs. Due to initiation of chondrogenic differentiation (Δt0-t7), 21 snoRNAs were differentially expressed (DE; FDRadj-p<0.05, logFC>1or<−1). Mineralization (Δt7-t14) induced DE of 23 snoRNAs. Comparing t0 with t14 resulted in DE of 43 snoRNAs. To anticipate on the biological relevance of DE snoRNAs, their rRNA target nucleotides were plotted in 18S, 5.8S and 28S rRNA secondary structures. This revealed that DE snoRNAs, amongst others, target nucleotide modifications in the 28S peptidyl transferase center and the 18S decoding center (DC). Snora40 was DE, targeting helix 27/18S rRNA. Helix 27 controls DC function. Helix 68 of 28S rRNA is part of the ribosome's E-site, therefore, DE snord36c and snora31 (targeting helix 68) could potentially fine-tune the translation mechanism. As a final example we found snord46 to be DE (target: helix 69/28S rRNA). Mutations in helix 69 have been shown to severely affect cell viability. Our data show that increased demand for translational capacity during chondrogenic differentiation is associated with differential expression of snoRNAs, potentially controlling ribosome fidelity via site-specific rRNA-modifications. These data enable us to determine the role of individual snoRNAs in tuning the chondrocyte's translational properties and current efforts focus on confirming site-specific rRNA-modifications and determine their biological relevance

To regenerate the complex tissue such as bone-cartilage construct using tissue engineering approaches, controllable differentiation of mesenchymal stem cells (BMSCs) into chondrogenic and osteogenic lineages is crucially important. Although bilayered scaffolds have been investigated in vitro and in vivo, no culture system is available to test BMSCs differentiation into bone and cartilage simultaneously in bilayered scaffolds. This study investigated a defined culture media, which supported osteoblast and chondrocyte differentiation depending on growth factors implemented in biomaterials. In 2-dimensional culture, BMSCs differentiated to chondrocytes when transforming growth factor-beta 3 (TGF-β3) was added to the defined media, whereas osteogenic differentiation was induced by adding bone morphogenetic protein 7 (BMP-7). BMSC differentiation to osteogenic and chondrogenic lineages was further strengthen in 3-dimensional culture. Proteoglycan formation, type II collagen, and aggrecan were upregulated in the defined media when BMSCs were mixed with fibrin gel impregnated with TGF-β3. Mineralization and the expression of osteogenic markers such as alkaline phosphatase, osteopontin, and osteoclacin were noticeable when BMSCs cultured in hydroxyapatite-tricalcium phosphate (HA/TCP) scaffolds coated with BMP-7. This study generated and tested a growth media, which could induce osteogenic and chondrogenic differentiation of BMSCs in one culture system. These results will help the development of tissue substitutes for multi-complexed tissues such as subchondral replacement

SBA-15 is a siliceous mesoporous ordered material with hexagonal arrangement of 9-nm tubular pores connected by micropores, high pore volume and abundance of silanol groups. This functionalised material could thus tailor the release kinetics of specific biomolecules to the clinical needs. Non-functionalized SBA-15 and its C8- or C3-alkyl-derivatives were coated with parathyroid hormone–related protein (PTHrP)(107–111) to assess their relative effects on osteoblastic cell growth and function. SBA-15 was functionalized with either octyl or propyl trimethoxysilane (C8 or C3 precursor, respectively) in ACN for 24h and then were coated (or not) by dipping in 10 nM PTHrP (107–111) solution for 24 h at 4°C. After air drying, biomaterials were transferred to culture dishes. MC3T3-E1 cells were cultured in differentiation medium with SBA-15, C3-SBA-15 and C8-SBA-15, loaded or not with the peptide. Cell viability and proliferation were evaluated by trypan blue exclusion and a proliferation kit (Promega), respectively. Alkaline phosphatase (ALP) activity and collagen secretion were determined by colorimetric methods. Gene expression was analyzed by real-time PCR. Mineralization was assessed by alizarin red staining. PTHrP(107–111)-coated SBA-15 increased cell proliferation (50%), cell viability (20%), and ALP activity (15%) over control values within 2–4 days. At day 2, collagen secretion increased (20%), and also the gene expression of ALP, PTHrP, and VEGF, which normalized at day 8, in these cells. An increase (by 30–40%) in all of these parameters was induced by peptide-coated C3-SBA-15 at day 4. Similar stimulatory effects were also observed with PTHrP(107–111)-coated C8-SBA-15 but only at day 8. At day 10, collagen secretion slightly increased (10–15%), and also mineralization (30–40%) with both functionalized materials coated with the PTHrP peptide. In conclusion, PTHrP(107–111)-coated SBA-15 stimulates osteoblastic function in vitro; an effect delayed by C3- or C8-functionalization. These data further support the clinical impact of this bioceramic as functionalized implants in vivo

Aims

The “2 to 10% strain rule” for fracture healing has been widely interpreted to mean that interfragmentary strain greater than 10% predisposes a fracture to nonunion. This interpretation focuses on the gap-closing strain (axial micromotion divided by gap size), ignoring the region around the gap where osteogenesis typically initiates. The aim of this study was to measure gap-closing and 3D interfragmentary strains in plated ovine osteotomies and associate local strain conditions with callus mineralization.