CAR (CARSKNKDC) is a systemically administered wound-homing peptide that specifically recognizes angiogenic blood vessels and extravasates into sites of injury. CAR peptide requires heparan sulfate proteoglycans (HSPGs) for its cell penetrating activity. Syndecan-4 (SDC4) is a HSPG and binding to it triggers the wound re-epithelialization process. We have discovered that CAR peptide has the inherent ability to promote wound healing; wounds close and re-epithelialize significantly faster in CAR treated mice than in control groups (PBS and mutant peptide, i.e. mCAR injections). To delineate the molecular mechanism by which CAR accelerates wound healing, we focused on the requirement of HSPG binding for CAR peptide function. We demonstrate that CAR peptide endocytosis and its stimulation of keratinocyte cell migration are both dependent on SDC4. Finally, we show that the systemic administration of CAR peptide stimulates wound re-epithelialization only in WT mice, but not in SDC4 knockout (KO) mice. As SDC4 has very restricted expression in skin wounds, we propose that CAR peptide activates SDC4 function to promote re-epithelialization. CAR peptide may provide an entirely new way of enhancing wound healing, and perhaps tissue regeneration in general. This therapeutic approach is systemic, yet target organ- and cell- specific, and dependent on the naturally occurring SDC4 dependent migratory pathway that is crucial for tissue regeneration

The purpose of this study was to develop a novel, minimally invasive therapy for nucleus pulposus augmentation without the need for major surgical incision. Two optimum patented self-assembling peptides based on natural amino acids were mixed with glycosaminoglycans (GAGs) to form reversible, tunable hydrogels that mimic the vital biological osmotic pumping action and aid in swelling pressure of the intervertebral disc (IVD). Separate peptide and GAG solutions can be switched from fluid to gel upon mixing inside the body. The gels were analysed using a series of complementary techniques (FTIR, TEM & rheometry) to determine their cross-length scale structure and properties. Approaches to developing a clinical product were then developed including the incorporation of a fluorescent probe and a CT contrast agents to aid visualization of the gels, and a semi-automatic syringe driver rig, incorporating a pressure sensor, for the delivery of the solutions into the intervertebral discs. The efficacy of the procedure in restoring disc height and biomechanics was examined using chemically degenerated bovine caudal samples. It was found the presence of the GAGs stabilized the peptides forming stiffer gels, even upon injection through a long (∼10cm) small gauge needle. The injected gels were easily visualized post injection by microCT and by eye during dissection under visible and UV light. It was also noted that following injection, the disc height of the degenerated samples was restored to a similar level of that observed for native discs. A hydrogel has been developed that is injected through a narrow bore needle using a semi-automatic delivery rig and forms a self-assembled gel in situ which has shown to restore the disc height. Further tests are now underway to examine their biomechanical performance across more physiological time periods

Abstract

Segmental bone transport (SBT) using an external fixator is currently a standard treatment for large-diameter bone defects at the donor site with low morbidity. However, long-term application of the device is needed for bone healing. In addition, patients who received SBT treatment sometimes fail to show bone repair and union at the docking site, and require secondary surgery. The objective of this study was to investigate whether a single injection of recombinant human bone morphogenetic protein 2 (rhBMP-2)-loaded artificial collagen-like peptide gel (rhBMP-2/ACG) accelerates consolidation and bone union at the docking site in a mouse SBT model. Six-month-old C57BL/6J mice were reconstructed by SBT with external fixator that has transport unit, and a 2.0-mm bone defect was created in the right femur. Mice were divided randomly into four treatment groups with eight mice in each group, Group CONT (immobile control), Group 0.2mm/d, Group 1.0mm/d, and Group BMP-2. Mice in Group 0.2mm/d and 1.0mm/d, bone segment was moved 0.2 mm per day for 10 days and 1.0 mm per day for 2 days, respectively. Mice in Group BMP-2 received an injection of 2.0 μg of rhBMP-2 dissolved in ACG into the bone defect site immediately after the defect-creating surgery and the bone segment was moved 1.0 mm/day for 2 days. All animals were sacrificed at eight weeks after surgery. Consolidation at bone defect site and bone union at docking site were evaluated radiologically and histologically. At the bone defect site, seven of eight mice in Group 0.2mm/d and two of eight mice in Group 1.0mm/d showed bone union. In contrast, all mice in Group CONT showed non-union at the bone defect site. At the docking site, four of eight mice in Group 0.2 mm/d and three of eight mice in Group 1.0 mm/d showed non-union. Meanwhile, all mice in Group BMP-2 showed bone union at the bone defect and docking sites. Bone volume and bone mineral content were significantly higher in Group 0.2mm/d and Group BMP-2 than in Group CONT. HE staining of tissue from Group 0.2mm/d and Group BMP-2 showed large amounts of longitudinal trabecular bone and regenerative new bone at eight weeks after surgery at the bone defect site. Meanwhile, in Group CONT and Group 1.0mm/d, maturation of regenerative bone at the bone defect site was poor. Differences between groups were analyzed using one-way ANOVA and a subsequent Bonferroni's post-hoc comparisons test. P < 0.05 was considered significant. rhBMP-2/ACG combined with SBT may be effective for enhancing bone healing in large bone defects without the need for secondary procedures

There is no diagnostic, non-invasive method for the early detection of loosening after total hip arthroplasty. In a pilot study, we have analysed two serum markers of bone remodelling, procollagen I C-terminal extension peptide (PICP) and cross-linked N-terminal telopeptide (NTx), as well as the diagnostic performance of NTx for the assessment of osteolysis. We recruited 21 patients with loosening (group I), 18 with a well-fixed prosthesis (group II) and 17 at the time of primary arthroplasty for osteoarthritis (OA) (group III). Internal normal reference ranges were obtained from 30 healthy subjects (group IV). The serum PICP level was found to be significantly lower in patients with OA and those with loosening, when compared with those with stable implants, while the NTx level was significantly increased only in the group with loosening, suggesting that collagen degradation depended on the altered bone turnover induced by the implant. This hypothesis was reinforced by the finding that the values in the pre-surgery patients and stable subjects were comparable with the reference range of younger healthy subjects. A high specificity and positive predictive value for NTx provided good diagnostic evidence of agreement between the test and the clinical and radiological evaluations. The NTx level could be used to indicate stability of the implant. However, further prospective, larger studies are necessary

Little is known on how sensory nerves and osteoclasts affect degenerative processes in subchondral bone in osteoarthritis (OA). Substance P (SP) effects on bone are ambivalent but physiological levels are critical for proper bone quality whereas α-calcitonin gene-related peptide (αCGRP) has anabolic effects. Here, we aimed to analyse the influence of an altered sensory neuropeptide microenvironment on subchondral bone in murine OA. Transection of the medial meniscotibial ligament (DMM) of the right hind leg induced joint instability leading to development of OA. Subchondral bone of tibiae from wildtype (WT), alendronate-treated WT (ALN, osteoclast inhibition), αCGRP- and SP- (Tachykinin (Tac)1) knockout mice was analysed by micro-computed tomography 4 and 12 weeks after DMM or sham surgery. Bone resorption marker CTX-I was measured in serum. We observed osteophytosis in all DMM groups and ALN sham mice 4 weeks after surgery but also in sham groups 12 weeks after surgery. In subchondral bone, bone volume density (BV/TV) increased from 4 to 12 weeks after surgery in DMM WT and Tac1-/− mice. DMM WT mice additionally had increased trabecular numbers (Tb.N.) and decreased trabecular space (Tb.Sp.) over time. Sham mice also showed time-dependent alterations in subchondral bone. In sham WT and αCGRP-/− mice specific bone surface (BS/BV) decreased and trabecular thickness (Tb.Th.) increased from 4 to 12 weeks after surgery while subchondral BV/TV of αCGRP-/− mice increased. Comparison of subchondral bone parameters at each time point showed elevated BV/TV in ALN DMM compared to WT DMM mice 4 weeks after surgery. In addition, both ALN sham and DMM mice showed a reduced BS/BV compared to WT. 4 weeks after sham surgery Tb.Th. was highest in ALN mice. In DMM WT mice Tb.Sp. was higher compared to ALN and αCGRP-/−. 12 weeks after surgery (late OA stage), BS/BV of ALN sham mice was significantly reduced in relation to ALN DMM, WT and Tac1-/− sham, while Tb.Th. increased compared to WT. DMM significantly decreased Tb.N. and increased Tb.Sp. in Tac1-/− compared to sham 12 weeks after surgery. CTX-I concentrations were significantly higher in ALN compared to Tac1-/− mice 4 weeks after sham surgery. 12 weeks after sham surgery CTX-I concentrations of WT mice were increased compared to αCGRP-/− and Tac1-/− mice. Over time, DMM induced stronger changes in subchondral bone of WT mice compared to knockout strains. WT and αCGRP-/− sham mice also show alterations in bone parameters over time indicating age-related effects on bone structure. SP deficiency enhanced DMM-induced structural bone alterations in late stage OA emphasizing the importance of SP under pathophysiological conditions. Osteoclast inhibition with alendronate proved to be preservative for time-dependent changes of subchondral bone observed in both, DMM and sham mice. Interestingly, ALN treatment did not reduce bone turnover marker CTX-I, and additionally promoted early osteophyte formation in sham mice

Introduction. Parathyroid hormone-related peptide (PTHrP) has been shown to be an important regulator of bone remodelling1. The aim of this study was to investigate the effect of the N-terminal domain of PTHrP (1–36) on osteogenic and angiogenic gene expression in human osteoblasts (HOB) and human bone marrow stromal cells (hBMSCs). Materials and Methods. Primary hBMSC's and HOBs were cultured in standard or osteogenic media with different concentrations of PTHrP, either continuously for 8, 24, 48 h and 9 days, or with 3 cycles of intermittent exposure (24 h with PTHrP, 24 h without) over 6 days. Cell lysates were then processed for analysis of gene expression. Expression of the osteogenic markers runt-related transcription factor 2 (RUNX-2), alkaline phosphatase (ALP) and Collagen 1, and the angiogenic marker; vascular endothelial growth factor (VEGF), were measured. Results. Exposure to PTHrP for ≤ 48 hours resulted in an upregulation of the angiogenic marker VEGF and the osteogenic markers RUNX-2, ALP and Collagen 1 in both cell types, peaking at a 1 nM PTHrP. Conversely, continuous exposure for 9 days, resulted in a downregulation of all osteogenic and angiogenic gene expression. HOB cells exposed intermittently to PTHrP showed an upregulation in VEGF and ALP, peaking at 10nM PTHrP. Discussion and Conclusion. Continuous exposure for short durations (<48 hours) and intermittent exposures of both HOB cells and BMSC's to PTHrP upregulated both osteogenic and angiogenic gene expression. Continuous exposure to 9 days however had the opposite effect, with a downregulation in gene transcription

Previously, we have demonstrated reduced biomechanical bone strength and matrix quality in Tachykinin (Tac)1-deficient mice lacking the sensory neuropeptide substance P (SP). A similar distortion of bone microarchitecture was described for α-calcitonin gene-related pepide (α-CGRP)-deficient mice. In previous studies we observed alterations in cell survival and differentiation capacity of bone cells isolated from wildtype mice when stimulated with SP and α-CGRP. We assume that changes in sensory neurotransmitter balance modulate bone cell metabolism thereby possibly contributing to inferior bone quality. In order to explore this hypothesis, we investigated and compared metabolic parameters in osteoblasts and osteoclasts isolated from SP- and α-CGRP-deficient mice and wildtype (WT) controls.

Bone marrow-derived macrophages (BMMs) and osteoblast-like cells from female C57Bl/6J (WT-control), Tac1-deficient (Tac1-/−) and α-CGRP-deficient (α-CGRP-/−) mice were isolated and differentiated according to established protocols (Niedermair et al., 2014). Cell metabolism studies were performed for enzyme activity and cell survival.

We observed reduced numbers of BMM from Tac1-/− and α-CGRP-/− mice after initial seeding compared to WT but no changes in viability. Osteoblast-like cells from Tac1-/− mice tend to migrate out faster from bone chips compared to WT-controls whereas migration of osteoblast-like cells from α-CGRP-/− mice was not affected. Osteoblasts and osteoclast/BMM cultures from WT mice endogenously synthesize and secrete SP as well as α-CGRP at a picomolar range. We found no changes regarding BMM or osteoblast proliferation from both, Tac1-/− and α-CGRP-/− mice when compared to WT-controls. Caspase 3/7-activity was reduced by trend in osteoclast/BMM cultures of α-CGRP-/− mice and significantly reduced in osteoclast/BMM cultures of Tac1-/− mice compared to WT-controls. We found significantly higher Caspase 3/7-activity in osteoblasts of Tac1-/− mice after 14 days of osteogenic culture conditions when compared to WT-controls whereas osteoblasts of α-CGRP-/− mice were unaffected. Cathepsin K enzyme activity was significantly reduced in osteoclast/BMM cultures of Tac1-/− and α-CGRP-/− mice compared to WT-controls. ALP activity of Tac1-/− osteoblasts was higher after 7 days and reduced after 21 days of osteogenic culture compared to WT-controls whereas ALP activity of osteoblasts of α-CGRP-/− mice was unchanged.

Acccording to our in vitro observations, we suggest some reduction in bone resorption rate but concomitantly a reduction in bone formation rate in Tac1-/− mice compared to WT-controls resulting in a net bone loss in these mice as bone resorption is faster than bone formation. Furthermore, we assume that bone resorption rate is slightly reduced in α-CGRP-/− mice but bone formation rate seems to be unchanged. Therefore we hypothesize that additional conditions present in vivo might contribute to the inferior bone properties of α-CGRP-/− mice.

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)

Orthopedic Device-Related Infections (ODRIs) are a major medical challenge, particularly due to the involvement of biofilm-encased and multidrug-resistant bacteria. Current treatments, based on antibiotic administration, have proven to be ineffective. Consequently, there is a need for antibiotic-free alternatives. Antimicrobial peptides (AMPs) are a promising solution due to their broad-spectrum of activity, high efficacy at very low concentrations, and low propensity to induce resistance. We aim to develop a new AMP-based chitosan nanogel to be injected during orthopedic device implantation to prevent ODRIs. Chitosan was functionalized with norbornenes (NorChit) through the reaction with carbic anhydride and then, a cysteine-modified AMP, Dhvar5, a peptide with potent antibacterial activity, even against methicillin-resistant Staphylococcus aureus (MRSA), was covalently conjugated to NorChit (NorChit- Dhvar5), through a thiol-norbornene photoclick chemistry (UV= 365 nm). For NorChit-Dhvar5 nanogels production, the NorChit-Dhvar5 solution (0.15% w/v) and Milli-Q water were injected separately into microfluidic system. The nanogels were characterized regarding size, concentration, and shape, using Transmission Electron Microscopy (TEM), Nanoparticle Tracking Analysis (NTA) and Dynamic light scattering (DLS). The nanogels antibacterial properties were assessed in Phosphate Buffer (PBS) for 6 h, against four relevant microorganisms (Pseudomonas aeruginosa, S. aureus and MRSA, and in Muller- Hinton Broth (MHB), 50% (v/v) in PBS, supplemented with human plasma (1% (v/v)), for 6 and 24 h against MRSA. The obtained NorChit-Dhvar5 nanogels, presented a round-shaped and ∼100 nm. NorChit- Dhvar5 nanogels in a concentration of 10. 10. nanogels/mL in PBS were capable of reducing the initial inoculum of P. aeruginosa by 99%, S. aureus by 99%, and MRSA by 90%. These results were corroborated by a 99% MRSA reduction, after 24 h in medium. Furthermore, NorChit-Dhvar5 nanogels do not demonstrate signs of cytotoxicity against MC3T3-E1 cells (a pre-osteoblast cell line) after 14 days, having high potential to prevent antibiotic-resistant infection in the context of ODRIs

Osteoarthritis (OA) is the most common degenerative joint disease causing joint immobility and chronic pain. Treatment is mainly based on alleviating pain and reducing disease progression. During OA progression the chondrocyte undergoes a hypertrophic switch in which extracellular matrix (ECM) -degrading enzymes are released, actively degrading the ECM. However, cell biological based therapies to slow down or reverse this katabolic phenotype are still to be developed. Bone morphogenetic protein 7 (BMP-7) has been shown to have OA disease-modifying properties. BMP-7 suppresses the chondrocyte hypertrophic and katabolic phenotype and may be the first biological treatment to target the chondrocyte phenotype in OA. However, intra-articular use of BMP-7 is at risk in the proteolytic and hydrolytic joint-environment. Weekly intra-articular injections are necessary to maintain biological activity, a frequency unacceptable for clinical use. Additionally, production of GMP-grade BMP-7 is challenging and expensive. To enable its clinical use, we sought for BMP-7 mimicking peptides better compatible with the joint-environment while still biologically active and which potentially can be incorporated in a drug-delivery system. We hypothesized that human BMP-7 derived peptides are able to mimic the disease modifying properties of the full-length human BMP-7 protein on the OA chondrocyte phenotype. A BMP-7 peptide library was synthesized consisting of overlapping 20-mer peptides with 18 amino-acids overlap between sequential peptides. OA human articular chondrocytes (HACs) were isolated from OA cartilage from total knee arthroplasty (n=18 donors). HACs were exposed to BMP-7 (1 nM) or BMP-7 library peptides at different concentrations (1, 10, 100 or 1000 nM). Gene-expression levels of important chondrogenic-, hypertrophic-, cartilage degrading- and inflammatory mediators were determined by RT-qPCR. GAG and ALP activity were determined using a colorimetric assay and PGE levels were measured by EIA. During the BMP-7 peptide library screening human BMP-7 derived peptides were screened for their full-length human BMP-7 mimicking properties at different concentrations (1, 10, 100 or 1000nM) on a pool of human chondrocytes. Gene expression as well as GAG, ALP and PGE2 level analysis revealed two distinct peptide regions in the BMP-7 protein based on their pro-chondrogenic and anti-OA phenotype actions on human OA chondrocytes. The two most promising peptides were further analysed for their OA chondrocyte disease modifying properties in the presence of OA synovial fluid, showing similar OA phenotype suppressive activity. Conclusively, we successfully identified two peptide regions in the BMP-7 protein with in vitro OA suppressive actions. Further biochemical fine-tuning of the peptides, and in vivo evaluation, will potentially result in the first peptide-based experimental OA treatment, addressing the hypertrophic and katabolic chondrocyte phenotype in OA

Osteoarthritis (OA) is a common age-related degenerative joint disease, affecting 7% of the global population, more than 500 million people worldwide. Exosomes from mesenchymal stem cells (MSCs) showed promise for OA treatment, but the insufficient biological targeting weakens its efficacy and might bring side effects. Here, we report the chondrocyte-targeted exosomes synthesized via click chemistry as a novel treatment for OA. Exosomes are isolated from human umbilical cord-derived MSCs (hUC-MSCs) using multistep ultracentrifugation process, and identified by electron microscope and nanoparticle tracking analysis (NTA). Chondrocyte affinity peptide (CAP) is conjugated on the surface of exosomes using click chemistry. For tracking, nontagged exosomes and CAP-exosomes are labeled by Dil, a fluorescent dye that highlights the lipid membrane of exosomes. To verify the effects of CAP-exosomes, nontagged exosomes and CAP-exosomes are added into the culture medium of interleukin (IL)-1β-induced chondrocytes. Immunofluorescence are used to test the expression of matrix metalloproteinase (MMP)-13. CAP-exosomes, compared with nontagged exosomes, are more easily absorbed by chondrocytes. What's more, CAP-exosomes induced lower MMP-13 expression of chondrocytes when compared with nontagged exosomes (p<0.001). CAP-exosomes show chondrocyte-targeting and exert better protective effect than nontagged exosomes on chondrocyte extracellular matrix. Histological and in vivo validation are now being conducted

Critical size bone defects are frequently caused by accidental trauma, oncologic surgery, and infection. Distraction osteogenesis (DO) is a useful technique to promote the repair of critical size bone defects. However, DO is usually a lengthy treatment, therefore accompanied with increased risks of complications such as infections and delayed union. Herein, we developed an innovative intramedullary biodegradable magnesium (Mg) nail to accelerate bone regeneration in critical size bone defect repair during DO. We observed that Mg nail induced almost 4-fold increase of new bone formation and over 5-fold of new vessel formation at 2 weeks after distraction. Mg nail upregulated the expression of calcitonin gene-related peptide (CGRP) in the new bone as compared with the DO alone group. We further revealed that blockade of the sensory nerve by overdose capsaicin blunted Mg nail enhanced critical size bone defect repair during the DO process. Moreover, inhibitors/antagonist of CGRP receptor, FAK, and VEGF receptor blocked the Mg nail stimulated vessel and bone formation. In summary, we revealed, for the first time, a CGRP-FAK-VEGF signaling axis linking sensory nerve and endothelial cells, which may be the main mechanism underlying Mg-enhanced critical size bone defect repair when combined with DO, suggesting a great potential of Mg implants in reducing DO treatment time for clinical applications

Introduction. The objective of the work is construction of a multi-bioactive scaffold based on that allows a space/time control over the regeneration of damaged bones by Medication-Related Osteonecrosis of the Jaw using a minimal invasive approach based on the injection of the fast-degrading pro neuro and angiogenic ELR (Elastin-Like Recombinamers) based hydrogels. Method. Chemical crosslinking facilitated the creation of multi-bioactive scaffolds using ELRs with reactive groups. Cell-loaded multi-bioactive scaffolds, prepared and incubated, underwent evaluation for adhesion, proliferation, angiogenic, and neurogenic potential. In vitro assessments utilized immunofluorescence staining and ELISA assays, while live-recorded monitoring and live-dead analysis ensured cytocompatibility. In rat and rabbit models, preformed scaffolds were subcutaneously implanted, and the regenerative process was evaluated over time. Rabbit models with MRONJ underwent traditional or percutaneous implantation, with histological evaluation following established bone histological techniques. Result. A 3D scaffold using ELR that combines various peptides with different degradation rates to guide both angiogenesis and neurogenesis has been developed. Notably, scaffolds with different degradation rates promoted distinct patterns of vascularization and innervation, facilitating integration with host tissue. This work demonstrates the potential for tailored tissue engineering, where the scaffold's bioactivities and degradation rates can control angiogenesis and neurogenesis. In an animal model of medication-related osteonecrosis of the jaw (MRONJ), the scaffold showed promising results in promoting bone regeneration in a necrotic environment, as confirmed by histological and imaging analyses. This study opens avenues for novel tissue-engineering strategies where precise control over vascularization and nerve growth is crucial. Conclusion. A groundbreaking dual approach, simultaneously targeting angiogenesis and innervation, addresses the necrotic bone in MRONJ syndrome. Vascularization and nerve formation play pivotal roles in driving reparative elements for bone regeneration. The scaffold achieves effective time/space control over necrotic bone regeneration. The authors are grateful for funding from the Spanish Government (PID2020-118669RA-I00)

The optimal treatment strategy for post-traumatic long bone non-unions is subject of an ongoing discussion. At the Maastricht University Medical Center (MUMC+) the induced membrane technique is used to treat post-traumatic long bone non-unions. This technique uses a multimodal treatment algorithm involving bone marrow aspirate concentrate (BMAC), the reamer-irrigator-aspirator (RIA) and P-15 bioactive peptide (iFactor, Cerapedics). Bioactive glass (S53P4 BAG, Bonalive) is added when infection is suspected. This study aims to objectify the effect of this treatment algorithm on the health-related quality of life (HRQoL) of patients with post-traumatic long bone non-unions. We hypothesized that HRQoL would improve after treatment. From January 2020 to March 2023, consecutive patients who were referred to a multidisciplinary (trauma, orthopaedic and plastic surgery) non-union clinic at the MUMC+, The Netherlands, were evaluated using the Non-Union Scoring System (NUSS). The EQ-5D-5L questionnaire and the Lower Extremity Functional Scale (LEFS) were employed to obtain HRQoL outcomes both prior to and subsequent to surgery, with a follow-up at 6, 18 and 35 weeks. Seventy-six patients were assessed at baseline (T0), with a mean NUSS of 40 (± 13 SD). Thirty-eight patients had their first follow-up, six weeks after surgery (T1). Thirty-one patients had a second follow-up at 18 weeks (T2), and twenty patients had the third follow-up at 35 weeks (T3). The EQ-5D index mean at baseline was 0.480, followed by an index of 0.618 at T1, 0.636 at T2, and 0.702 at T3. A significant difference was found in the HRQoL score between T0 and T1, as well as T2 and T3 (p<0.001; p=0.011). The mean LEFS significantly increased from 26 before intervention to 34, 39, and 43 after treatment (p<0.001; p=0.033; p=0.016). This study demonstrated a significant improvement in the health-related quality of life of patients with post-traumatic long bone non-unions after the standardized treatment algorithm following the induced membrane technique

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

Introduction. Osteoarthritis (OA) causes pain, stiffness, and loss of function due to degenerative changes in joint cartilage and bone. In some forms of OA, exercise can alleviate symptoms by improving joint mobility and stability. However, excessive training after joint injury may have negative consequences for OA development. Sensory nerve fibers in joints release neuropeptides like alpha-calcitonin gene-related peptide (alpha-CGRP), potentially affecting OA progression. This study investigates the role of alpha-CGRP in OA pathogenesis under different exercise regimen in mice. Method. OA was induced in C57Bl/6J WT mice and alpha-CGRP KO mice via surgical destabilization of the medial meniscus (DMM) at 12 weeks of age (N=6). Treadmill exercise began 2 weeks post-surgery and was performed for 30 minutes, 5 days a week, for 2 or 6 weeks at intense (16 m/min, 15° incline) or moderate (10 m/min, 5° incline) levels. Histomorphometric assessment of cartilage degradation (OARSI scoring), serum cytokine analysis, immunohistochemistry, and nanoCT analysis were conducted. Result. OARSI scoring confirmed OA induction 4 weeks post-DMM surgery, with forced exercise exacerbating cartilage degradation regardless of intensity. No significant genotype-dependent differences were observed. Serum analysis revealed elevated cytokine levels associated with OA and inflammation in KO mice compared to WT mice 4 and 8 weeks post-surgery (VEGF-A, MCP-1, CXCL10, RANTES, MIP1-alpha, MIP1-beta, and RANKL). The observed effects were often exacerbated by intense exercise but rarely by DMM surgery. NanoCT analysis demonstrated increased sclerotic bone changes after 6 weeks of forced exercise in KO mice compared to WT mice. Conclusion. Our results suggest an OA promoting effect of exercise in early disease stages of posttraumatic OA. Intense exercise induced inflammatory processes correlated to increased cytokine levels in the serum that might exacerbate OA pathogenesis in later stages. The neuropeptide alpha-CGRP might play a role in protecting against these adverse effects

Cell-based tissue engineering is a promising approach for treating cartilage lesions but the optimal cell-scaffold combination for hyaline cartilage regeneration has yet to be identified. Novel hydrogels allow including tailored tissue type specific modifications with physiologically relevant peptides, by this selectively influencing the cell response. Aim of this study was to modify a poly(ethylene glycol) (PEG)/heparin hydrogel by functionalization with cell instructive peptides introducing matrix-metalloprotease (MMP)-degradability, the cell adhesion motif RGD, or collagen binding motifs (CKLER, CWYRGRL) to improve cartilage matrix deposition in tissue engineering constructs. The hydrogels were formed by mixing thiol-endfunctionalized (MMP-insensitive) starPEG or starPEG-MMP-conjugates carrying MMP-sensitive peptides at every arm and maleimide-functionalized heparin [1] in the presence or absence of cell instructive peptides. Human mesenchymal stromal cells (MSC) or porcine chondrocytes were grown in the hydrogels for up to 4 weeks in vitro under chondrogenic conditions, and in vivo in subcutaneous pockets of immunodeficient mice. MMP-sensitive and –insensitive starPEG/heparin hydrogels supported chondrogenic differentiation of MSC according to induction of COL2A1, BGN and ACAN mRNA expression. Enhanced MMP-sensitivity and therefore degradability increased cell viability and proliferation. RGD-modification of the hydrogels induced cell-spreading and an intensively interconnected cell network. Other than hypothesized, CKLER and CWYRGRL were unable to raise collagen deposition in constructs in vitro. Matrix deposition in chondrocyte-containing peptide-functionalized hydrogels was high and the instructive effect of the hydrogels on chondrocytes appeared stronger in vivo where the merely pericellular cartilaginous matrix deposition was overcome in RGD-functionalized starPEG/heparin hydrogels. Peptide-functionalized starPEG/heparin hydrogel altered cell morphology, proliferation and differentiation with MSC being similar sensitive to cell-matrix interaction peptides like articular chondrocytes. We also demonstrated that in vivoperformance of cell instructive hydrogels can exceed results gained by in vitromodels. Altogether, the manipulation of hydrogel constructs with signaling cues is considered promising for functional cartilage tissue engineering

Objectives. The molecular mechanism of rheumatoid arthritis (RA) remains elusive. We conducted a protein-protein interaction network-based integrative analysis of genome-wide association studies (GWAS) and gene expression profiles of RA. Methods. We first performed a dense search of RA-associated gene modules by integrating a large GWAS meta-analysis dataset (containing 5539 RA patients and 20 169 healthy controls), protein interaction network and gene expression profiles of RA synovium and peripheral blood mononuclear cells (PBMCs). Gene ontology (GO) enrichment analysis was conducted by DAVID. The protein association networks of gene modules were generated by STRING. Results. For RA synovium, the top-ranked gene module is HLA-A, containing TAP2, HLA-A, HLA-C, TAPBP and LILRB1 genes. For RA PBMCs, the top-ranked gene module is GRB7, consisting of HLA-DRB5, HLA-DRA, GRB7, CD63 and KIT genes. Functional enrichment analysis identified three significant GO terms for RA synovium, including antigen processing and presentation of peptide antigen via major histocompatibility complex class I (false discovery rate (FDR) = 4.86 × 10 – 4), antigen processing and presentation of peptide antigen (FDR = 2.33 × 10 – 3) and eukaryotic translation initiation factor 4F complex (FDR = 2.52 × 10 – 2). Conclusion. This study reported several RA-associated gene modules and their functional association networks. Cite this article: X. Xiao, J. Hao, Y. Wen, W. Wang, X. Guo, F. Zhang. Genome-wide association studies and gene expression profiles of rheumatoid arthritis: an analysis. Bone Joint Res 2016;5:314–319. DOI: 10.1302/2046-3758.57.2000502

One of the core tenets of our philosophy for tissue regeneration include the use of “raw materials,” where biomaterials themselves serve as both building blocks and bioactive signals. In recent years, a few groups around the world have gravitated toward cartilage matrix as a potentially chondroinductive material for cartilage regeneration. The major challenge to date in cartilage injury has been creating a biomaterial-only strategy that is capable of regenerating true hyaline-like cartilage without the addition of growth factors or exogenous cells. In the past few years, we have focused our efforts on establishing chondroinductivity in vitro, and in developing new materials synthesis strategies to provide ease of application for orthopedic surgeons in the operating room. By leveraging nanotechnology, we have developed a paste-like material constructed from cartilage matrix with encouraging mechanical performance post-crosslinking, and which avoids contraction after extended time. Looking to the future, we are working on next-generation approaches to chondroinductive materials. We have encouraging preliminary data which suggest the possibility of a chondroinductive response to a novel peptide sequence in vitro, which may be enhanced by simultaneous inclusion of adhesion peptides. Initial in vivo data in regeneration of rabbit femoral condyle cartilage defects may suggest promising regenerative capabilities with hydrogels based on these peptides. If indeed chondroinductive materials exist, and if they can be delivered easily, are safe, and can be provided at reasonable cost and with a reasonable regulatory strategy, chondroinductive materials may hold the potential to revolutionize cartilage regeneration