Advanced therapies in orthopaedics.

Advanced therapies are expected to play a crucial role in supporting repair after injury, halting the degeneration of musculoskeletal tissue to enable and promote physical activity. Despite advancements, the progress in developing advanced therapies in orthopaedics lags behind specialties like oncology, since innovative regenerative treatment strategies fall short of their expectations in musculoskeletal clinical trials. Researchers should focus on understanding the mechanism of action behind the investigated target before conducting clinical trials. Strategic research networks are needed that not only enhance scientific exchange among like-minded researchers but need to include early on commercial views, companies and venture perspectives, regulatory insights and reimbursement perspectives. Only in such collaborations essential roadblocks towards clinical trials and go-to-patients be overcome.

Long-termin vitromaintenance of plasma cells in a hydrogel-enclosed human bone marrow microphysiological 3D model system.

Plasma cells (PCs) in bone marrow (BM) play an important role in both protective and pathogenic humoral immune responses, e.g. in various malignant and non-malignant diseases such as multiple myeloma, primary and secondary immunodeficiencies and autoimmune diseases. Dedicated microenvironmental niches in the BM provide PCs with biomechanical and soluble factors that support their long-term survival. There is a high need for appropriate and robust model systems to better understand PCs biology, to develop new therapeutic strategies for PCs-related diseases and perform targeted preclinical studies with high predictive value. Most preclinical data have been derived fromin vivostudies in mice, asin vitrostudies of human PCs are limited due to restricted survival and functionality in conventional 2D cultures that do not reflect the unique niche architecture of the BM. We have developed a microphysiological, dynamic 3D BM culture system (BM-MPS) based on human primary tissue (femoral biopsies), mechanically supported by a hydrogel scaffold casing. While a bioinert agarose casing did not support PCs survival, a photo-crosslinked collagen-hyaluronic acid (Col-HA) hydrogel preserved the native BM niche architecture and allowed PCs survivalin vitrofor up to 2 weeks. Further, the Col-HA hydrogel was permissive to lymphocyte migration into the microphysiological system´s circulation. Long-term PCs survival was related to the stable presence in the culture of soluble factors, as APRIL, BAFF, and IL-6. Increasing immunoglobulins concentrations in the medium confirm their functionality over culture time. To the best of our knowledge, this study is the first report of successful long-term maintenance of primary-derived non-malignant PCsin vitro. Our innovative model system is suitable for in-depthin vitrostudies of human PCs regulation and exploration of targeted therapeutic approaches such as CAR-T cell therapy or biologics.

Volumetric evaluation of osteotomy gap following mandibular bilateral sagittal split osteotomy using a novel semi-automated approach: a pilot study.

OBJECTIVES: To establish an analysis pipeline for the volumetric evaluation of the osteotomy site after bilateral sagittal split osteotomy (BSSO). PATIENTS AND METHODS: Cone-beam computed tomography (CBCT) was performed before, directly after BSSO, and 6-12 months after surgery. Image segmentations of each osteotomy gap data set were performed manually by four physicians and were compared to a semi-automatic segmentation approach. RESULTS: Five patients with a total of ten osteotomy gaps were included. The mean interclass correlation coefficient (ICC) of individual patients was 0.782 and the standard deviation 0.080 when using the manual segmentation approach. However, the mean ICC of the evaluation of anatomical sites and time points separately was 0.214, suggesting a large range of deviation within the manual segmentation of each rater. The standard deviation was 0.355, further highlighting the extent of the variation. In contrast, the semi-automatic approach had a mean ICC of 0.491 and a standard deviation of 0.365, which suggests a relatively higher agreement among the operators compared to the manual segmentation approach. Furthermore, the volume of the osteotomy gap in the semi-automatic approach showed the same tendency in every site as the manual segmentation approach, but with less deviation. CONCLUSION: The semi-automatic approach developed in the present study proved to be valid as a standardised method with high repeatability. Such image analysis methods could help to quantify the progression of bone healing after BSSO and beyond, eventually facilitating the earlier identification of patients with retarded healing.

In Vitro Transcribed mRNA Immunogenicity Induces Chemokine-Mediated Lymphocyte Recruitment and Can Be Gradually Tailored by Uridine Modification.

Beyond SARS-CoV2 vaccines, mRNA drugs are being explored to overcome today's greatest healthcare burdens, including cancer and cardiovascular disease. Synthetic mRNA triggers immune responses in transfected cells, which can be reduced by chemically modified nucleotides. However, the side effects of mRNA-triggered immune activation on cell function and how different nucleotides, such as the N1-methylpseudouridine (m1Ψ) used in SARS-CoV2 vaccines, can modulate cellular responses is not fully understood. Here, cellular responses toward a library of uridine-modified mRNAs are investigated in primary human cells. Targeted proteomics analyses reveal that unmodified mRNA induces a pro-inflammatory paracrine pattern marked by the secretion of chemokines, which recruit T and B lymphocytes toward transfected cells. Importantly, the magnitude of mRNA-induced changes in cell function varies quantitatively between unmodified, Ψ-, m1Ψ-, and 5moU-modified mRNA and can be gradually tailored, with implications for deliberately exploiting this effect in mRNA drug design. Indeed, both the immunosuppressive effect of stromal cells on T-cell proliferation, and the anti-inflammatory effect of IL-10 mRNA are enhanced by appropriate uridine modification. The results provide new insights into the effects of mRNA drugs on cell function and cell-cell communication and open new possibilities to tailor mRNA-triggered immune activation to the desired pro- or anti-inflammatory application.

The enhancer landscape predetermines the skeletal regeneration capacity of stromal cells.

Multipotent stromal cells are considered attractive sources for cell therapy and tissue engineering. Despite numerous experimental and clinical studies, broad application of stromal cell therapeutics is not yet emerging. A major challenge is the functional diversity of available cell sources. Here, we investigated the regenerative potential of clinically relevant human stromal cells from bone marrow (BMSCs), white adipose tissue, and umbilical cord compared with mature chondrocytes and skin fibroblasts in vitro and in vivo. Although all stromal cell types could express transcription factors related to endochondral ossification, only BMSCs formed cartilage discs in vitro that fully regenerated critical-size femoral defects after transplantation into mice. We identified cell type-specific epigenetic landscapes as the underlying molecular mechanism controlling transcriptional stromal differentiation networks. Binding sites of commonly expressed transcription factors in the enhancer and promoter regions of ossification-related genes, including Runt and bZIP families, were accessible only in BMSCs but not in extraskeletal stromal cells. This suggests an epigenetically predetermined differentiation potential depending on cell origin that allows common transcription factors to trigger distinct organ-specific transcriptional programs, facilitating forward selection of regeneration-competent cell sources. Last, we demonstrate that viable human BMSCs initiated defect healing through the secretion of osteopontin and contributed to transient mineralized bone hard callus formation after transplantation into immunodeficient mice, which was eventually replaced by murine recipient bone during final tissue remodeling.

Immuno-engineered mRNA combined with cell adhesive niche for synergistic modulation of the MSC secretome.

In vitro transcribed (IVT-)mRNA has entered center stage for vaccine development due to its immune co-stimulating properties. Given the widely demonstrated safety of IVT-mRNA-based vaccines, we aimed to adopt IVT-mRNA encoding VEGF for secretory phenotype modulation of therapeutic cells. However, we observed that the immunogenicity of IVT-mRNA impairs the endogenous secretion of pro-angiogenic mediators from transfected mesenchymal stromal cells, instead inducing anti-angiogenic chemokines. This inflammatory secretome modulation limits the application potential of unmodified IVT-mRNA for cell therapy manufacturing, pro-angiogenic therapy and regenerative medicine. To uncouple immunogenicity from the protein expression functionality, we immuno-engineered IVT-mRNA with different chemically modified ribonucleotides. 5-Methoxy-uridine-modification of IVT-mRNA rescued the endogenous secretome pattern of transfected cells and prolonged secretion of IVT-mRNA-encoded VEGF. We found that high secretion of IVT-mRNA-encoded protein further depends on optimized cell adhesion. Cell encapsulation in a collagen-hyaluronic acid hydrogel increased secretion of IVT-mRNA-encoded VEGF and augmented the endogenous secretion of supporting pro-angiogenic mediators, such as HGF. Integrating minimally immunogenic mRNA technology with predesigned matrix-derived cues allows for the synergistic combination of multiple dimensions of cell manipulation and opens routes for biomaterial-based delivery of mRNA-engineered cell products. Such multimodal systems could present a more biologically relevant way to therapeutically address complex multifactorial processes such as tissue ischemia, angiogenesis, and regeneration.

Individual immune cell and cytokine profiles determine platelet-rich plasma composition.

OBJECTIVE: Platelet-rich plasma (PRP) therapy is increasingly popular to treat musculoskeletal diseases, including tendinopathies and osteoarthritis (OA). To date, it remains unclear to which extent PRP compositions are determined by the immune cell and cytokine profile of individuals or by the preparation method. To investigate this, we compared leukocyte and cytokine distributions of different PRP products to donor blood samples and assessed the effect of pro-inflammatory cytokines on chondrocytes. DESIGN: For each of three PRP preparations (ACP®, Angel™, and nSTRIDE® APS), products were derived using whole blood samples from twelve healthy donors. The cellular composition of PRP products was analyzed by flow cytometry using DURAClone antibody panels (DURAClone IM Phenotyping Basic and DURAClone IM T Cell Subsets). The MESO QuickPlex SQ 120 system was used to assess cytokine profiles (V-PLEX Proinflammatory Panel 1 Human Kit, Meso Scale Discovery). Primary human chondrocyte 2D and 3D in vitro cultures were exposed to recombinant IFN-γ and TNF-α. Proliferation and chondrogenic differentiation were quantitatively assessed. RESULTS: All three PRP products showed elevated portions of leukocytes compared to baseline levels in donor blood. Furthermore, the pro-inflammatory cytokines IFN-γ and TNF-α were significantly increased in nSTRIDE® APS samples compared to donor blood and other PRP products. The characteristics of all other cytokines and immune cells from the donor blood, including pro-inflammatory T cell subsets, were maintained in all PRP products. Chondrocyte proliferation was impaired by IFN-γ and enhanced by TNF-α treatment. Differentiation and cartilage formation were compromised upon treatment with both cytokines, resulting in altered messenger ribonucleic acid (mRNA) expression of collagen type 1A1 (COL1A1), COL2A1, and aggrecan (ACAN) as well as reduced proteoglycan content. CONCLUSIONS: Individuals with elevated levels of cells with pro-inflammatory properties maintain these in the final PRP products. The concentration of pro-inflammatory cytokines strongly varies between PRP products. These observations may help to unravel the previously described heterogeneous response to PRP in OA therapy, especially as IFN-γ and TNF-α impacted primary chondrocyte proliferation and their characteristic gene expression profile. Both the individual's immune profile and the concentration method appear to impact the final PRP product. TRIAL REGISTRATION: This study was prospectively registered in the Deutsches Register Klinischer Studien (DRKS) on 4 November 2021 (registration number DRKS00026175).

Chondrocyte Isolation from Loose Bodies-An Option for Reducing Donor Site Morbidity for Autologous Chondrocyte Implantation.

Loose bodies (LBs) from patients with osteochondritis dissecans (OCD) are usually removed and discarded during surgical treatment of the defect. In this study, we address the question of whether these LBs contain sufficient viable and functional chondrocytes that could serve as a source for autologous chondrocyte implantation (ACI) and how the required prolonged in vitro expansion affects their phenotype. Chondrocytes were isolated from LBs of 18 patients and compared with control chondrocyte from non-weight-bearing joint regions (n = 7) and bone marrow mesenchymal stromal cells (BMSCs, n = 6) obtained during primary arthroplasty. No significant differences in the initial cell yield per isolation and the expression of the chondrocyte progenitor cell markers CD44 + /CD146+ were found between chondrocyte populations from LBs (LB-CH) and control patients (Ctrl-CH). During long-term expansion, LB-CH exhibited comparable viability and proliferation rates to control cells and no ultimate cell cycle arrest was observed within 12 passages respectively 15.3 ± 1.1 mean cumulative populations doublings (CPD). The chondrogenic differentiation potential was comparable between LB-CH and Ctrl-CH, but both groups showed a significantly higher ability to form a hyaline cartilage matrix in vitro than BMSC. Our data suggest that LBs are a promising cell source for obtaining qualitatively and quantitatively suitable chondrocytes for therapeutic applications, thereby circumventing donor site morbidity as a consequence of the biopsies required for the current ACI procedure.

Synchrotron-based characterization of arthroprosthetic CoCrMo particles in human bone marrow.

Particles released from cobalt-chromium-molybdenum (CoCrMo) alloys are considered common elicitors of chronic inflammatory adverse effects. There is a lack of data demonstrating particle numbers, size distribution and elemental composition of bone marrow resident particles which would allow for implementation of clinically relevant test strategies in bone marrow models at different degrees of exposure. The aim of this study was to investigate metal particle exposure in human periprosthetic bone marrow of three types of arthroplasty implants. Periprosthetic bone marrow sections from eight patients exposed to CoCrMo particles were analyzed via spatially resolved and synchrotron-based nanoscopic X-ray fluorescence imaging. These analyses revealed lognormal particle size distribution patterns predominantly towards the nanoscale. Analyses of particle numbers and normalization to bone marrow volume and bone marrow cell number indicated particle concentrations of up to 1 × 1011 particles/ml bone marrow or 2 × 104 particles/bone marrow cell, respectively. Analyses of elemental ratios of CoCrMo particles showed that particularly the particles' Co content depends on particle size. The obtained data point towards Co release from arthroprosthetic particles in the course of dealloying and degradation processes of larger particles within periprosthetic bone marrow. This is the first study providing data based on metal particle analyses to be used for future in vitro and in vivo studies of possible toxic effects in human bone marrow following exposure to arthroprosthetic CoCrMo particles of different concentration, size, and elemental composition. Graphical abstract.

Epigenetic lockdown of CDKN1A (p21) and CDKN2A (p16) characterises the neoplastic spindle cell component of giant cell tumours of bone.

Giant cell tumour of bone (GCTB) comprises the eponymous osteoclastic multinucleated giant cells eliciting bone lysis, an H3F3A-mutated neoplastic mononucleated fibroblast-like cell population, and H3F3A wild-type mononucleated stromal cells. In this study, we characterised four new cell lines from GCTB. Furthermore, we compared the genome-wide DNA methylation profile of 13 such tumours and three further cell lines with giant cell-rich lesions comprising three H3F3B-mutated chondroblastomas, three USP6-rearranged aneurysmal bone cysts, three non-ossifying fibromas, two hyperparathyroidism-associated brown tumours as well as mesenchymal stem cells, osteoblasts, and osteoclasts. In an unsupervised analysis, we delineated GCTB and chondroblastomas from the other analysed tumour entities. Using comparative methylation analysis, we demonstrated that the methylation pattern of the cell lines approximately equals that of H3F3A-mutated stromal cells in tissue. These patterns more resemble that of osteoblasts than that of mesenchymal stem cells, which argues for the osteoblast as the cell of origin of giant cell tumours of bone. Using enrichment analysis, we detected distinct hypermethylated clusters containing histone and collagen genes as well as target genes of the tumour suppressor p53. We found that the promotor regions of CDKN1A, CDKN2A, and IGFBP3 are methylated more strongly in GCTB than in the other giant cell-containing lesions, mesenchymal stem cells, osteoblasts, and osteoclasts (p < 0.001). This hypermethylation correlates with the lower gene expression at the mRNA level for these three genes in the cell lines, the lack of p16 and p21 in these cell lines, and the lower expression of p16 and p21 in GCTB. Overall, our analysis reveals characteristic DNA methylation patterns of giant cell tumours of bone and chondroblastomas and shows that cell lines of giant cell tumours of bone are a valid model for further analysis of H3F3A-mutated tumour cells. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Batch Effects during Human Bone Marrow Stromal Cell Propagation Prevail Donor Variation and Culture Duration: Impact on Genotype, Phenotype and Function.

Donor variation is a prominent critical issue limiting the applicability of cell-based therapies. We hypothesized that batch effects during propagation of bone marrow stromal cells (BMSCs) in human platelet lysate (hPL), replacing fetal bovine serum (FBS), can affect phenotypic and functional variability. We therefore investigated the impact of donor variation, hPL- vs. FBS-driven propagation and exhaustive proliferation, on BMSC epigenome, transcriptome, phenotype, coagulation risk and osteochondral regenerative function. Notably, propagation in hPL significantly increased BMSC proliferation, created significantly different gene expression trajectories and distinct surface marker signatures, already after just one passage. We confirmed significantly declining proliferative potential in FBS-expanded BMSC after proliferative challenge. Flow cytometry verified the canonical fibroblastic phenotype in culture-expanded BMSCs. We observed limited effects on DNA methylation, preferentially in FBS-driven cultures, irrespective of culture duration. The clotting risk increased over culture time. Moreover, expansion in xenogenic serum resulted in significant loss of function during 3D cartilage disk formation and significantly increased clotting risk. Superior chondrogenic function under hPL-conditions was maintained over culture. The platelet blood group and isoagglutinins had minor impact on BMSC function. These data demonstrate pronounced batch effects on BMSC transcriptome, phenotype and function due to serum factors, partly outcompeting donor variation after just one culture passage.

Bio-instructive hydrogel expands the paracrine potency of mesenchymal stem cells.

The therapeutic efficacy of clinically applied mesenchymal stromal cells (MSCs) is limited due to their injection into harshin vivoenvironments, resulting in the significant loss of their secretory function upon transplantation. A potential strategy for preserving their full therapeutic potential is encapsulation of MSCs in a specialized protective microenvironment, for example hydrogels. However, commonly used injectable hydrogels for cell delivery fail to provide the bio-instructive cues needed to sustain and stimulate cellular therapeutic functions. Here we introduce a customizable collagen I-hyaluronic acid (COL-HA)-based hydrogel platform for the encapsulation of MSCs. Cells encapsulated within COL-HA showed a significant expansion of their secretory profile compared to MSCs cultured in standard (2D) cell culture dishes or encapsulated in other hydrogels. Functionalization of the COL-HA backbone with thiol-modified glycoproteins such as laminin led to further changes in the paracrine profile of MSCs. In depth profiling of more than 250 proteins revealed an expanded secretion profile of proangiogenic, neuroprotective and immunomodulatory paracrine factors in COL-HA-encapsulated MSCs with a predicted augmented pro-angiogenic potential. This was confirmed by increased capillary network formation of endothelial cells stimulated by conditioned media from COL-HA-encapsulated MSCs. Our findings suggest that encapsulation of therapeutic cells in a protective COL-HA hydrogel layer provides the necessary bio-instructive cues to maintain and direct their therapeutic potential. Our customizable hydrogel combines bioactivity and clinically applicable properties such as injectability, on-demand polymerization and tissue-specific elasticity, all features that will support and improve the ability to successfully deliver functional MSCs into patients.

H3F3A-mutated giant cell tumour of bone without giant cells-clinical presentation, radiology and histology of three cases.

AIMS: Giant cell tumour of bone (GCTB) is histologically defined as a lesion containing reactive giant cells and a neoplastic mononuclear cell population; in up to 92% of cases, GCTB is characterised by a specific mutation of the histone gene H3F3A. The cellular composition ranges from giant-cell-rich to giant-cell-poor. The diagnosis of GCTB can be challenging, and several other lesions need to be excluded, e.g. aneurysmal bone cysts, non-ossifying fibromas, chondroblastomas, brown tumours, and giant-cell-rich osteosarcomas. Our aim was to analyse the clinical history, imaging, molecular pathology and histology of three H3F3A-mutated bone tumours without detectable giant cells. None of the patients received denosumab therapy. METHODS AND RESULTS: Diagnostic material was obtained by curettage or resection and/or biopsy. Common histomorphological features of all three reported lesions were fibrocytic, oval cells in a background of osteoid and an absence of multinuclear giant cells as confirmed with CD68 immunohistochemistry. We used immunohistochemistry and Sanger sequencing to demonstrate positivity for the H3.3 p.G34W mutation. Differential diagnoses were systematically excluded on the basis of histomorphology, immunohistochemistry, and fluorescence in-situ hybridisation. The imaging (radiography, computed tomography, and magnetic resonance imaging) for all three cases is presented and discussed. CONCLUSIONS: We believe that these GCTBs without giant cells expand one end of the heterogeneous range of GCTB. Because of the lack of giant cells, correct diagnosis of GCTB is challenging or even impossible on histological grounds alone. In these cases, detection of the characteristic H3F3A mutation (G34W-specific antibody RM263 or sequencing) is extremely helpful for diagnosing those lesions without giant cells as giant cell tumours of bone.

Metal-Specific Biomaterial Accumulation in Human Peri-Implant Bone and Bone Marrow.

Metallic implants are frequently used in medicine to support and replace degenerated tissues. Implant loosening due to particle exposure remains a major cause for revision arthroplasty. The exact role of metal debris in sterile peri-implant inflammation is controversial, as it remains unclear whether and how metals chemically alter and potentially accumulate behind an insulating peri-implant membrane, in the adjacent bone and bone marrow (BM). An intensively focused and bright synchrotron X-ray beam allows for spatially resolving the multi-elemental composition of peri-implant tissues from patients undergoing revision surgery. In peri-implant BM, particulate cobalt (Co) is exclusively co-localized with chromium (Cr), non-particulate Cr accumulates in the BM matrix. Particles consisting of Co and Cr contain less Co than bulk alloy, which indicates a pronounced dissolution capacity. Particulate titanium (Ti) is abundant in the BM and analyzed Ti nanoparticles predominantly consist of titanium dioxide in the anatase crystal phase. Co and Cr but not Ti integrate into peri-implant bone trabeculae. The characteristic of Cr to accumulate in the intertrabecular matrix and trabecular bone is reproducible in a human 3D in vitro model. This study illustrates the importance of updating the view on long-term consequences of biomaterial usage and reveals toxicokinetics within highly sensitive organs.

MSC Therapies for COVID-19: Importance of Patient Coagulopathy, Thromboprophylaxis, Cell Product Quality and Mode of Delivery for Treatment Safety and Efficacy.

Numerous clinical trials of mesenchymal stromal/stem cells (MSCs) as a new treatment for coronavirus-induced disease (COVID-19) have been registered recently, most of them based on intravenous (IV) infusion. There is no approved effective therapy for COVID-19, but MSC therapies have shown first promise in the treatment of acute respiratory distress syndrome (ARDS) pneumonia, inflammation, and sepsis, which are among the leading causes of mortality in COVID-19 patients. Many of the critically ill COVID-19 patients are in a hypercoagulable procoagulant state and at high risk for disseminated intravascular coagulation, thromboembolism, and thrombotic multi-organ failure, another cause of high fatality. It is not yet clear whether IV infusion is a safe and effective route of MSC delivery in COVID-19, since MSC-based products express variable levels of highly procoagulant tissue factor (TF/CD142), compromising the cells' hemocompatibility and safety profile. Of concern, IV infusions of poorly characterized MSC products with unchecked (high) TF/CD142 expression could trigger blood clotting in COVID-19 and other vulnerable patient populations and further promote the risk for thromboembolism. In contrast, well-characterized products with robust manufacturing procedures and optimized modes of clinical delivery hold great promise for ameliorating COVID-19 by exerting their beneficial immunomodulatory effects, inducing tissue repair and organ protection. While the need for MSC therapy in COVID-19 is apparent, integrating both innate and adaptive immune compatibility testing into the current guidelines for cell, tissue, and organ transplantation is critical for safe and effective therapies. It is paramount to only use well-characterized, safe MSCs even in the most urgent and experimental treatments. We here propose three steps to mitigate the risk for these vulnerable patients: (1) updated clinical guidelines for cell and tissue transplantation, (2) updated minimal criteria for characterization of cellular therapeutics, and (3) updated cell therapy routines reflecting specific patient needs.

Mesenchymal stromal cell and bone marrow concentrate therapies for musculoskeletal indications: a concise review of current literature.

The interest on applying mesenchymal stromal cells (MSCs) in orthopedic disorders has risen tremendously in the last years due to scientific successes in preclinical in vitro and animal model studies. In a wide range of diseases and injuries of the musculoskeletal system, MSCs are currently under evaluation, but so far have found access to clinical use only in few cases. The current assignment is to translate the acquired knowledge into clinical practice. Therefore, this review aims at presenting a synopsis of the up-to-date status of the use of MSCs and MSC related cell products in musculoskeletal indications. Clinical studies were included, whereas preclinical and animal study data not have been considered. Most studies published so far investigate the final outcome applying bone marrow derived MSCs. In fewer trials the use of adipose tissue derived MSCs and allogenic MSCs was investigated in different applications. Although the reported results are equivocal in the current literature, the vast majority of the studies shows a benefit of MSC based therapies depending on the cell sources and the indication in clinical use. In summary, the clinical use of MSCs in patients in orthopedic indications has been found to be safe. Standardized protocols and clear definitions of the mechanisms of action and the mode and timing of application as well as further coordinated research efforts will be necessary for finally adding MSC based therapies in standard operating procedures and guidelines for the clinicians treating orthopedic disorders.

Adult Osteosclerotic Metaphyseal Dysplasia With Progressive Osteonecrosis of the Jaws and Abnormal Bone Resorption Pattern Due to a LRRK1 Splice Site Mutation.

Osteosclerotic metaphyseal dysplasia (OSMD) is a rare autosomal recessive sclerosing skeletal dysplasia. We report on a 34-year-old patient with sandwich vertebrae, platyspondyly, osteosclerosis of the tubular bones, pathologic fractures, and anemia. In the third decade, he developed osteonecrosis of the jaws, which was progressive in spite of repeated surgical treatment over a period of 11 years. An iliac crest bone biopsy revealed the presence of hypermineralized cartilage remnants, large multinucleated osteoclasts with abnormal morphology, and inadequate bone resorption typical for osteoclast-rich osteopetrosis. After exclusion of mutations in TCIRG1 and CLCN7 we performed trio-based exome sequencing. The novel homozygous splice-site mutation c.261G>A in the gene LRRK1 was found and co-segregated with the phenotype in the family. cDNA sequencing showed nearly complete skipping of exon 3 leading to a frameshift (p.Ala34Profs*33). Osteoclasts differentiated from the patient's peripheral blood monocytes were extremely large. Instead of resorption pits these cells were only capable of superficial erosion. Phosphorylation of L-plastin at position Ser5 was strongly reduced in patient-derived osteoclasts showing a loss of function of the mutated LRRK1 kinase protein. Our analysis indicates a strong overlap of LRRK1-related OSMD with other forms of intermediate osteopetrosis, but an exceptional abnormality of osteoclast resorption. Like in other osteoclast pathologies an increased risk for progressive osteonecrosis of the jaws should be considered in OSMD, an intermediate form of osteopetrosis. © 2020 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research.

Evaluation of 3D Printed Gelatin-Based Scaffolds with Varying Pore Size for MSC-Based Adipose Tissue Engineering.

Adipose tissue engineering aims to provide solutions to patients who require tissue reconstruction following mastectomies or other soft tissue trauma. Mesenchymal stromal cells (MSCs) robustly differentiate into the adipogenic lineage and are attractive candidates for adipose tissue engineering. This work investigates whether pore size modulates adipogenic differentiation of MSCs toward identifying optimal scaffold pore size and whether pore size modulates spatial infiltration of adipogenically differentiated cells. To assess this, extrusion-based 3D printing is used to fabricate photo-crosslinkable gelatin-based scaffolds with pore sizes in the range of 200-600 µm. The adipogenic differentiation of MSCs seeded onto these scaffolds is evaluated and robust lipid droplet formation is observed across all scaffold groups as early as after day 6 of culture. Expression of adipogenic genes on scaffolds increases significantly over time, compared to TCP controls. Furthermore, it is found that the spatial distribution of cells is dependent on the scaffold pore size, with larger pores leading to a more uniform spatial distribution of adipogenically differentiated cells. Overall, these data provide first insights into the role of scaffold pore size on MSC-based adipogenic differentiation and contribute toward the rational design of biomaterials for adipose tissue engineering in 3D volumetric spaces.

Niche-mimicking interactions in peptide-functionalized 3D hydrogels amplify mesenchymal stromal cell paracrine effects.

Cells encounter complex environments in vivo where they interact with the extracellular matrix, neighboring cells, and soluble cues, which together influence their fate and function. However, the interplay of these interactions and their collective impact on the regenerative effects of mesenchymal stromal cells (MSCs) remains insufficiently explored. Here, we show that 3D culture in microporous (~125 µm) hydrogels that passively promote cell-cell interactions sensitizes MSCs to growth factors, particularly to IGF-1. IGF-1 enhances MSC paracrine secretion activity, and application of secreted factors to myoblasts potently stimulates their migration and differentiation. In contrast, the paracrine activity of MSCs encapsulated in nanoporous (~10 nm) hydrogels remain unchanged. Blocking N-cadherin on MSCs abrogates the stimulatory effects of IGF-1 in microporous but not nanoporous hydrogels. The role of N-cadherin in regulating MSC function is further clarified by functionalizing alginates with the HAVDI peptide sequence that is derived from the extracellular domain of N-cadherin and that acts to mimic cell-cell interactions. MSCs encapsulated in nanoporous HAVDI-gels, but not in gels functionalized with a scrambled sequence, show heightened paracrine activity in response to IGF-1. These findings reveal how interactions with the matrix, neighboring cells, and soluble factors impact and maximize the regenerative potential of MSCs.