Local pH is stated to acidify after bone fracture. However, the time course and degree of acidification remain unknown. Whether the acidification pattern within a fracture hematoma is applicable to adjacent muscle hematoma or is exclusive to this regenerative tissue has not been studied to date. Thus, in this study, we aimed to unravel the extent and pattern of acidification in vivo during the early phase post musculoskeletal injury. Local pH changes after fracture and muscle trauma were measured simultaneously in two pre-clinical animal models (sheep/rats) immediately after and up to 48 h post injury. The rat fracture hematoma was further analyzed histologically and metabolomically. In vivo pH measurements in bone and muscle hematoma revealed a local acidification in both animal models, yielding mean pH values in rats of 6.69 and 6.89, with pronounced intra- and inter-individual differences. The metabolomic analysis of the hematomas indicated a link between reduction in tricarboxylic acid cycle activity and pH, thus, metabolic activity within the injured tissues could be causative for the different pH values. The significant acidification within the early musculoskeletal hematoma could enable the employment of the pH for novel, sought-after treatments that allow for spatially and temporally controlled drug release.

There is a growing need for scaffold material with tissue-specific bioactivity for use in regenerative medicine, tissue engineering, and for surgical repair of structural defects. We developed a novel composite biomaterial by processing human cardiac extracellular matrix (ECM) into a hydrogel and combining it with cell-free amniotic membrane via a dry-coating procedure. Cardiac biocompatibility and immunogenicity were tested in vitro using human cardiac fibroblasts, epicardial progenitor cells, murine HL-1 cells, and human immune cells derived from buffy coat. Processing of the ECM preserved important matrix proteins as demonstrated by mass spectrometry. ECM coating did not alter the mechanical characteristics of decellularized amniotic membrane but did cause a clear increase in adhesion capacity, cell proliferation and viability. Activated monocytes secreted less pro-inflammatory cytokines, and both macrophage polarization towards the pro-inflammatory M1 type and T cell proliferation were prevented. We conclude that the incorporation of human cardiac ECM hydrogel shifts and enhances the bioactivity of decellularized amniotic membrane, facilitating its use in future cardiac applications.

Nutrient supply via a functional vasculature is essential during regenerative processes, tissue growth, and homeostasis. 3D bioprinting offers the opportunity to engineer vascularized constructs by combining cells and biocompatible materials in specifically designed fashions. However, the complexity of microvascular dynamic networks can hardly be recapitulated yet, even by sophisticated 3D manufacturing. Ideally, the natural organizational competences of endothelial cells will be harnessed such that engineered vascular networks self‐assemble to form complex, controllable microvascular patterns. Here, a bioengineering approach is presented to control microvascular structure formation and to steer cellular self‐assembly of endothelial and supporting cells within a multi‐material stereolithographic 3D bioprinting concept. Bioengineered vascularized constructs are generated by controlled cell deposition in an enzymatically degradable or a non‐degradable material. In vitro, the microvascular structures are regulated in distribution, network orientation, vessel length and branching behavior and developed lumen, signs of vascular stabilization and an interconnected vascular network including anastomosis. This novel biofabrication approach demonstrates the capability to control microvascular network formation by using cellular and spatial cues allowing the generation of distinctly yet precisely vascularized constructs. Such novel approach of controlled microvascular formation may play a fundamental role in the development of vascularized implants or in vitro screening models.

Bone morphogenetic protein-2 (BMP-2) is a known key mediator of physiological bone regeneration and is clinically approved for selected musculoskeletal interventions. Yet, broad usage of this growth factor is impeded due to side effects that are majorly evoked by high dosages and burst release kinetics. In this study, mesoporous bioactive glass microspheres (MBGs), produced by an aerosol-assisted spray-drying scalable process, were loaded with BMP-2 resulting in prolonged, low-dose BMP-2 release without affecting the material characteristics. In vitro, MBGs were found to be cytocompatible and to induce a pro-osteogenic response in primary human mesenchymal stromal cells (MSCs). In a pre-clinical rodent model, BMP-2 loaded MBGs significantly enhanced bone formation and influenced the microarchitecture of newly formed bone. The MBG carriers alone performed equal to the untreated (empty) control in most parameters tested, while additionally exerting mild pro-angiogenic effects. Using MBGs as a biocompatible, pro-regenerative carrier for local and sustained low dose BMP-2 release could limit side effects, thus enabling a safer usage of BMP-2 as a potent pro-osteogenic growth factor.

Regeneration of large bone defects remains a clinical challenge until today. While existing biomaterials are predominantly addressing bone healing via direct, intramembranous ossification (IO), bone tissue formation via a cartilage phase, so-called endochondral ossification (EO) has been shown to be a promising alternative strategy. However, pure biomaterial approaches for EO induction are sparse and the knowledge how material components can have bioactive contribution to the required cartilage formation is limited. Here, we combined a previously developed purely architecture-driven biomaterial approach with the release of therapeutic metal ions from tailored silicate microparticles. The delivery platform was free of calcium phosphates (CaP) that are known to support IO but not EO and was employed for the release of lithium (Li), magnesium (Mg), strontium (Sr) or zinc (Zn) ions. We identified an ion-specific cellular response in which certain metal ions strongly enhanced cell recruitment into the material and showed superior chondrogenesis and deposition collagen II by human mesenchymal stromal cells (MSCs). At the same time, microparticle incorporation in some cases altered the mechanical properties of the biomaterial with consequences for cell-induced biomaterial contraction and scaffold wall deformation. Collectively, the results suggest that the incorporation of metal-doped silicate microparticles has the potential to further improve the bioactivity of architectured biomaterials for bone defect healing via EO. STATEMENT OF SIGNIFICANCE: Endochondral bone healing, a process that resembles embryonic skeletal development, has gained prominence in regenerative medicine. However, most therapeutic biomaterial strategies are not optimized for endochondral bone healing but instead target direct bone formation through intramembranous ossification. Here, we report on a novel approach to accelerate biomaterial-guided endochondral bone healing by combining cell-guiding collagen scaffolds with therapeutic metal-doped silicate microparticles. While other strategies, such as hypoxia-mimic drugs and iron-chelating biomaterials, have been documented in the literature before to enhance endochondral ossification, our approach uniquely implements enhanced bioactivity into a previously developed biomaterial strategy for bone defect regeneration. Enhanced cell recruitment into the material and more pronounced chondrogenesis were observed for specific hybrid scaffold formulations, suggesting a high relevance of this new biomaterial for improved endochondral bone healing.

Despite years of diligent research in fracture healing, an unmet clinical need for safe and effective pharmacological treatments to improve bone regeneration persists with 10 – 20 % of fracture cases exhibiting impaired healing. Bone morphogenetic protein-2 (BMP-2) is a known key mediator of physiological bone regeneration and is clinically approved for selected musculoskeletal interventions. Yet, broad usage of this growth factor is impeded due to side effects that are majorly evoked by high dosages and burst release kinetics. In this study, mesoporous bioactive glass microspheres (MBGs) produced by an aerosol assisted spray-drying, scalable process were found to be biocompatible and to induce a pro-osteogenic response on human MSCs in vitro . Loading of the MBGs with BMP-2 resulted in prolonged, low-dose BMP-2 release without affecting the material features. In a pre-clinical rodent model, BMP-2 loaded MBGs significantly enhanced bone formation and influenced the microarchitecture of newly formed bone. The MBG carriers alone performed equal to the untreated (empty) control in most parameters tested, while additionally exerting mild pro-angiogenic effects. Using MBGs as a biocompatible, pro-regenerative carrier for local and sustained low dose BMP-2 release could limit side effects, thus enabling a safer usage of BMP-2 as a potent pro-osteogenic growth factor.