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Effective regeneration of bone defects often presents significant challenges, particularly in patients with decreased tissue regeneration capacity due to extensive trauma, disease, and/or advanced age. A number of studies have focused on enhancing bone regeneration by applying mesenchymal stromal cells (MSCs) or MSC-based bone tissue engineering strategies. However, translation of these approaches from basic research findings to clinical use has been hampered by the limited understanding of MSC therapeutic actions and complexities, as well as costs related to the manufacturing, regulatory approval, and clinical use of living cells and engineered tissues. More recently, a shift from the view of MSCs directly contributing to tissue regeneration toward appreciating MSCs as \"cell factories\" that secrete a variety of bioactive molecules and extracellular vesicles with trophic and immunomodulatory activities has steered research into new MSC-based, \"cell-free\" therapeutic modalities. The current review recapitulates recent developments, challenges, and future perspectives of these various MSC-based bone tissue engineering and regeneration strategies.

Cardiovascular diseases remain the leading cause of death worldwide; hence there is an increasing focus on developing physiologically relevant in vitro cardiovascular tissue models suitable for studying personalized medicine and pre-clinical tests. Despite recent advances, models that reproduce both tissue complexity and maturation are still limited. We have established a scaffold-free protocol to generate multicellular, beating human cardiac microtissues in vitro from hiPSCs—namely human organotypic cardiac microtissues (hOCMTs)—that show some degree of self-organization and can be cultured for long term. This is achieved by the differentiation of hiPSC in 2D monolayer culture towards cardiovascular lineage, followed by further aggregation on low-attachment culture dishes in 3D. The generated hOCMTs contain multiple cell types that physiologically compose the heart and beat without external stimuli for more than 100 days. We have shown that 3D hOCMTs display improved cardiac specification, survival and metabolic maturation as compared to standard monolayer cardiac differentiation. We also confirmed the functionality of hOCMTs by their response to cardioactive drugs in long-term culture. Furthermore, we demonstrated that they could be used to study chemotherapy-induced cardiotoxicity. Due to showing a tendency for self-organization, cellular heterogeneity, and functionality in our 3D microtissues over extended culture time, we could also confirm these constructs as human cardiac organoids (hCOs). This study could help to develop more physiologically-relevant cardiac tissue models, and represent a powerful platform for future translational research in cardiovascular biology.

An injectable hybrid hydrogel is synthesized, comprising silk fibroin (SF) and chondroitin sulfate (CS) through di‐tyrosine formation bond of SF chains. CS and SF are reported with excellent biocompatibility as tissue engineering scaffolds. Nonetheless, the rapid degradation rate of pure CS scaffolds presents a challenge to effectively recreate articular cartilage. As CS is one of the cartilage extracellular matrix (ECM) components, it has the potential to enhance the biological activity of SF‐based hydrogel in terms of cartilage repair. Therefore, altering the CS concentrations (i.e., 0 wt%, 0.25 wt%, 0.5 wt%, 1 wt%, and 2 wt%), which are interpenetrated between SF β‐sheets and chains, can potentially adjust the physical, chemical, and mechanical features of these hybrid hydrogels. The formation of β‐sheets by 30 days of immersion in de‐ionized (DI) water can improve the compression strength of the SF/CS hybrid hydrogels in comparison with the same SF/CS hybrid hydrogels in the dried state. Biological investigation and observation depicts proper cell attachment, proliferation and cell viability for C28/I2 cells. Gene expression of sex‐determining region YBox 9 (SOX9), Collagen II α1, and Aggrecan (AGG) exhibits positive C3H10T1/2 growth and expression of cartilage‐specific genes in the 0.25 wt% and 0.5 wt% SF/CS hydrogels. In the current study, an injectable hybrid hydrogel containing silk fibroin (SF) and chondroitin sulfate (CS) is prepared and characterized through di‐tyrosine formation bonds between SF chains using horse radish peroxide (HRP) and hydrogen peroxide (H2O2). In fact, altering CS amounts, which are interpenetrated between SF β‐sheets and chains adjusts the physical, chemical, and biological features of these hybrid hydrogels.

Summary Damage to cells and tissues is one of the driving forces of aging and age-related diseases. Various repair systems are in place to counteract this functional decline. In particular, the property of adult stem cells to self-renew and differentiate is essential for tissue homeostasis and regeneration. However, their functionality declines with age (Rando, 2006). One organ that is notably affected by the reduced differentiation capacity of stem cells with age is the skeleton. Here, we found that circulating microvesicles impact on the osteogenic differentiation capacity of mesenchymal stem cells in a donor-age-dependent way. While searching for factors mediating the inhibitory effect of elderly derived microvesicles on osteogenesis, we identified miR-31 as a crucial component. We demonstrated that miR-31 is present at elevated levels in the plasma of elderly and of osteoporosis patients. As a potential source of its secretion, we identified senescent endothelial cells, which are known to increase during aging in vivo (Erusalimsky, 2009). Endothelial miR-31 is secreted within senescent cell-derived microvesicles and taken up by mesenchymal stem cells where it inhibits osteogenic differentiation by knocking down its target Frizzled-3. Therefore, we suggest that microvesicular miR-31 in the plasma of elderly might play a role in the pathogenesis of age-related impaired bone formation and that miR-31 might be a valuable plasma-based biomarker for aging and for a systemic environment that does not favor cell-based therapies whenever osteogenesis is a limiting factor.

Spinal cord contusion injury leads to severe loss of gray and white matter and subsequent deficit of motor and sensory functions below the lesion. In this study, we investigated whether application of murine clonal embryonic neuroectodermal stem cells can prevent the spinal cord secondary damage and induce functional recovery. Stem cells (NE-GFP-4C cell line) were grafted intraspinally or intravenously immediately or one week after thoracic spinal cord contusion injury. Control animals received cell culture medium or fibrin intraspinally one week after injury. Functional tests (Basso, Beattie, Bresnahan, CatWalk®) and detailed morphological analysis were performed to evaluate the effects of grafted cells. Stem cells applied either locally or intravenously induced significantly improved functional recovery compared with their controls. Morphologically, stem cell grafting prevented the formation of secondary injury and promoted sparing of the gray and white matters. The transplanted cells integrated into the host tissue and differentiated into neurons, astrocytes, and oligodendrocytes. In intraspinally grafted animals, the corticospinal tract axons regenerated along the ventral border of the cavity and have grown several millimeters, even beyond the caudal end of the lesion. The extent of regeneration and functional improvement was inversely related to the amounts of chondroitin sulphate and ephrin-B2 molecules around the cavity and to the microglial and astrocytic reactions in the injured segment early after injury. The grafts produced glial cell derived neurotrophic factor, macrophage inflammatory protein-1a, interleukin (IL)-6 and IL-10 in a paracrine fashion for at least one week. Treating the grafted cords with neutralizing antibodies against these four factors through the use of osmotic pumps nearly completely abolished the effect of the graft. The non-significant functional improvement after function blocking is likely because the stem cell derivatives settled in the injured cord. These data suggest that grafted neuroectodermal stem cells are able to prevent the secondary spinal cord damage and induce significant regeneration via multiple mechanisms.

Low level light therapy receives increasing interest in the fields of tissue regeneration and wound healing. Several in vivo studies demonstrated the positive effects of LLLT on angiogenesis. This study aimed to investigate the underlying properties in vitro by comparing the effects of light therapy by light emitting diodes of different wavelengths on endothelial cells in vitro . Human umbilical vein endothelial cells were treated with either 475 nm, 516 nm or 635 nm light. Control cells were not illuminated. 2D proliferation was quantified by manual counting. HUVEC migration was analyzed by performing a 2D wound scratch assay and a 3D bead assay. The influence of LLLT on early vasculogenic events was determined in a 3D fibrin co-culture model with adipose-derived stem cells. Stimulation with both red and green pulsed LED light significantly increased HUVEC proliferation and 3D migration. Moreover, HUVEC showed increased 2D migration potential with green light stimulation. The treatment with blue light was ineffective. Several parameters showed that green light was even more potent to stimulate proliferation and migration of endothelial cells than clinically well-established red light therapy. Further studies have to focus on intracellular mechanisms induced by different wavelengths in order to optimize this promising therapy in tissue regeneration.

Pulsed electromagnetic field (PEMF) therapy is a non-invasive treatment that delivers electric and magnetic fields to tissues via inductive coils inducing salutary effects. Previously PEMF was reported to accelerate gas transport in the liquid phase. Drawing analogies from electrical elements, we hypothesized that PEMF can modulate transmembrane potential of biological membranes. Given that mitochondria interact with gaseous molecules such as oxygen and nitric oxide (NO) and contain voltage-sensitive channels, this study focused on the effects of a single PEMF device with a low input-energy and a 1ms, 30 kHz sine wave duty cycle per pulse on mitochondrial function. Experiments were conducted using cell cultures, tissue homogenates, and isolated mitochondria. We assessed mitochondrial membrane potential, NO levels, and mitochondrial respiration. We found no evidence that PEMF restores mitochondrial respiration inhibited by NO, but it can be restored by exposure of mitochondria to blue light. Our findings show that PEMF selectively stimulates respiration linked to ATP-synthesis affecting less uncoupled respiration. This suggests that changes in ATP-synthesis underlies the primary beneficial effects observed here. The underlying mechanisms may include interaction of PEMF with mitochondrial transport systems or activity of mitochondrial complexes. The exact mechanisms still should be investigated.

In the past decade, adipose tissue became a highly interesting source of adult stem cells for plastic surgery and regenerative medicine. The isolated stromal vascular fraction (SVF) is a heterogeneous cell population including the adipose-derived stromal/stem cells (ASC), which showed regenerative potential in several clinical studies and trials. SVF should be provided in a safe and reproducible manner in accordance with current good manufacturing practices (cGMP). To ensure highest possible safety for patients, a precisely defined procedure with a high-quality control is required. Hence, an increasing number of adipose tissue-derived cell isolation systems have been developed. These systems aim for a closed, sterile, and safe isolation process limiting donor variations, risk for contaminations, and unpredictability of the cell material. To isolate SVF from adipose tissue, enzymes such as collagenase are used. Alternatively, in order to avoid enzymes, isolation systems using physical forces are available. Here, we provide an overview of known existing enzymatic and non-enzymatic adipose tissue-derived cell isolation systems, which are patented, published, or already on the market.

Clinical trials of therapeutic angiogenesis by vascular endothelial growth factor (VEGF) gene delivery failed to show efficacy. Major challenges include the need to precisely control in vivo distribution of growth factor dose and duration of expression. Recombinant VEGF protein delivery could overcome these issues, but rapid in vivo clearance prevents the stabilization of induced angiogenesis. Here, we developed an optimized fibrin platform for controlled delivery of recombinant VEGF, to robustly induce normal, stable, and functional angiogenesis. Murine VEGF164 was fused to a sequence derived from α2-plasmin inhibitor (α2-Pl1–8) that is a substrate for the coagulation factor fXIIIa, to allow its covalent cross-linking into fibrin hydrogels and release only by enzymatic cleavage. An α2-Pl1–8–fused variant of the fibrinolysis inhibitor aprotinin was used to control the hydrogel degradation rate, which determines both the duration and effective dose of factor release. An optimized aprotinin-α2-Pl1–8 concentration ensured ideal degradation over 4 wk. Under these conditions, fibrin-α2-Pl1–8-VEGF164 allowed exquisitely dose-dependent angiogenesis: concentratios ≥25 μg/mL caused widespread aberrant vascular structures, but a 500-fold concentration range (0.01–5.0 μg/mL) induced exclusively normal, mature, nonleaky, and perfused capillaries, which were stable after 3 mo. Optimized delivery of fibrin-α2-Pl1–8-VEGF164 was therapeutically effective both in ischemic hind limb and wound-healing models, significantly improving angiogenesis, tissue perfusion, and healing rate. In conclusion, this optimized platform ensured (i) controlled and highly tunable delivery of VEGF protein in ischemic tissue and (ii) stable and functional angiogenesis without introducing genetic material and with a limited and controllable duration of treatment. These findings suggest a strategy to improve safety and efficacy of therapeutic angiogenesis.

Background The aim of this study was to evaluate potential synergistic effects of a single, local application of human umbilical cord MSC-derived sEVs in combination with a low dose of recombinant human rhBMP-2 to promote the regeneration of a metaphyseal femoral defect in an osteoporotic rat model. Methods 6 weeks after induction of osteoporosis by bilateral ventral ovariectomy and administration of a special diet, a total of 64 rats underwent a distal femoral metaphyseal osteotomy using a manual Gigli wire saw. Defects were stabilized with an adapted Y-shaped mini-locking plate and were subsequently treated with alginate only, or alginate loaded with hUC-MSC-sEVs (2 × 10 9 ), rhBMP-2 (1.5 µg), or a combination of sEVs and rhBMP-2 ( n  = 16 for each group). 6 weeks post-surgery, femora were evaluated by µCT, descriptive histology, and biomechanical testing. Results Native radiographs and µCT analysis confirmed superior bony union with callus formation after treatment with hUC-MSC-sEVs in combination with a low dose of rhBMP-2. This finding was further substantiated by histology, showing robust defect consolidation 6 weeks after treatment. Torsion testing of the explanted femora revealed increased stiffness after application of both, rhBMP-2 alone, or in combination with sEVs, whereas torque was only significantly increased after treatment with rhBMP-2 together with sEVs. Conclusion The present study demonstrates that the co-application of hUC-MSC-sEVs can improve the efficacy of rhBMP-2 to promote the regeneration of osteoporotic bone defects.