Several hemostatic strategies rely on the use of blood components such as fibrinogen and thrombin, which suffer from high cost and short shelf‐life. Here, a cost‐effective synthetic biomaterial is developed for rapid local hemostasis. Instead of using thrombin, thrombin‐receptor‐agonist‐peptide‐6 (TRAP6) is covalently engineered in polyvinyl alcohol (PVA) hydrogels. Soluble PVA‐TRAP6 is first prepared by covalent attachment of cysteine‐containing TRAP6 onto the backbone of PVA‐norbornenes (PVA‐NB) through photoconjugation. Cytotoxicity studies using C2C12 myoblasts indicate that PVA‐NB and PVA‐TRAP6 are nontoxic. Thromboelastography reveals that hemostatic activity of TRAP6 is retained in conjugated form, which is comparable to free TRAP6 solutions with equal concentrations. A 0.1% PVA‐TRAP6 solution can shorten the clotting time (CT) to ca. 45% of the physiological CT. High platelet‐activating efficiency is further confirmed by platelet aggregation assay and flow cytometry (FACS). For potential clinical applications, TRAP6‐presenting hydrogel particulates (PVA‐TRAP6‐P) are developed for local platelet activation and hemostasis. PVA‐TRAP6‐P is prepared by biofunctionalization of photopolymerized PVA‐NB hydrogel particulates (PVA‐NB‐P) with TRAP6. It is demonstrated that PVA‐TRAP6‐P can effectively shorten the CT to ca. 50%. FACS shows that PVA‐TRAP6‐P can activate platelets to a comparable extent as soluble TRAP6 control. Altogether, PVA‐TRAP6‐P represents a promising class of biomaterials for safe hemostasis and wound healing.

A dual thermoresponsive and magnetic colloidal gel matrix is described for enhanced stem‐cell culture. The combined properties of the material allow enzyme‐free passaging and expansion of mesenchymal stem cells, as well as isolation of cells postculture by the simple process of lowering the temperature and applying an external magnetic field. The colloidal gel can be reconfigured with thermal and magnetic stimuli to allow patterning of cells in discrete zones and to control movement of cells within the porous matrix during culture.

Nanocrystalline hydroxyapatite (HA) has good biocompatibility and the potential to support bone formation. It represents a promising alternative to autologous bone grafting, which is considered the current gold standard for the treatment of low weight bearing bone defects. The purpose of this study was to compare three bone substitute pastes of different HA content and particle size with autologous bone and empty defects, at two time points (6 and 12 months) in an ovine scapula drillhole model using micro-CT, histology and histomorphometry evaluation. The nHA-LC (38% HA content) paste supported bone formation with a high defect bridging-rate. Compared to nHA-LC, Ostim® (35% HA content) showed less and smaller particle agglomerates but also a reduced defect bridging-rate due to its fast degradation The highly concentrated nHA-HC paste (48% HA content) formed oversized particle agglomerates which supported the defect bridging but left little space for bone formation in the defect site. Interestingly, the gold standard treatment of the defect site with autologous bone tissue did not improve bone formation or defect bridging compared to the empty control. We concluded that the material resorption and bone formation was highly impacted by the particle-specific agglomeration behaviour in this study.

BackgroundThe need for bone graft substitutes including those being developed to be applied together with new strategies of bone regeneration such as tissue engineering and cell-based approaches is growing. No large animal model of bone regeneration has been accepted as a standard testing model. Standardization may be the key to moving systematically towards better bone regeneration. This study aimed to establish a model of bone regeneration in the sheep that lends itself to strict standardization and in which a number of substances can be tested within the same animal. To this end the caudal border of the ovine scapula was used as a consistent bed of mineralized tissue that provided sufficient room for a serial alignment of multiple experimental drill holes.ResultsThe findings show that for the sake of standardization, surgery should be restricted to the middle part of the caudal margin, an area at least 80 mm proximal from the Glenoid cavity, but not more than 140 mm away from it, in the adult female Land Merino sheep. A distance of 5 mm from the caudal margin should also be observed.ConclusionsThis standardized model with defined uniform defects and defect sites results in predictable and reproducible bone regeneration processes. Defects are placed unilaterally in only one limb of the animal, avoiding morbidity in multiple limbs. The fact that five defects per animal can be evaluated is conducive to intra-animal comparisons and reduces the number of animals that have to be subject to experimentation.

Thermoreversible hydrogels for tissue engineering (TE) purposes have gained increased attention in recent years as they can be combined with cells and drugs and directly injected into the body. Following the fate of transplanted cells in situ is essential in characterizing their distribution and survival, as well as the expression of specific markers or cell-matrix interactions. Existing histological embedding methods, such as paraffin wax embedding, can mechanically damage some biomaterials during processing. In this study, we describe a broadly applicable preparation protocol that allows the handling of delicate, thermoreversible scaffolds for histological sectioning. The gelatin solution permits the embedding of samples at 37 °C, which suits the solid phase of most TE scaffolds. A thermoreversible scaffold of polycaprolactone microparticles, combined with poly(polyethylene glycol methacrylate ethyl ether) and containing human adipose-derived stem cells, was prepared for histology by an initial gelatin embedding step in addition to the standard cryosectioning and paraffin processing protocols. Sections were evaluated by hematoxylin eosin staining and immunostaining for human vimentin. The gelatin embedding retained the scaffold particles and permitted the complete transfer of the construct. After rapid cooling, the solid gelatin blocks could be cryosectioned and paraffin infiltrated. In contrast to direct cryosectioning or paraffin infiltration, the extended protocol preserved the scaffold structure as well as the relevant cell epitopes, which subsequently allowed for immunostaining of human cells within the material. The gelatin embedding method proposed is a generalizable alternative to standard preparations for histological examination of a variety of delicate samples.

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Mesenchymal stem cells (MSCs) have the potential to repair and regenerate damaged tissues in response to injury, such as fracture or other tissue injury. Bone marrow and adipose tissue are the major sources of MSCs. Previous studies suggested that the regenerative activity of stem cells can be enhanced by exposure to tissue microenvironments. The aim of our project was to investigate whether extracellular matrix (ECM) engineered from human induced pluripotent stem cells-derived mesenchymal-like progenitors (hiPSCs-MPs) can enhance the regenerative potential of human bone marrow mesenchymal stromal cells (hBMSCs).ECM was engineered from hiPSC-MPs. ECM structure and composition were characterized before and after decellularization using immunofluorescence and biochemical assays. hBMSCs were cultured on the engineered ECM, and differentiated into osteogenic, chondrogenic and adipogenic lineages. Growth and differentiation responses were compared to tissue culture plastic controls.Decellularization of ECM resulted in efficient cell elimination, as observed in our previous studies. Cultivation hBMSCs on the ECM in osteogenic medium significantly increased hBMSC growth, collagen deposition and alkaline phosphatase activity. Furthermore, expression of osteogenic genes and matrix mineralization were significantly higher compared to plastic controls. Chondrogenic micromass culture on the ECM significantly increased cell growth and expression of chondrogenic markers, including glycosaminoglycans and collagen type II. Adipogenic differentiation of hBMSCs on the ECM resulted in significantly increased hBMSC growth, but significantly reduced lipid vacuole deposition compared to plastic controls. Together, our studies suggest that BMSCs differentiation into osteogenic and chondrogenic lineages can be enhanced, whereas adipogenic activity is decreased by the culture on engineered ECM. Contribution of specific matrix components and underlying mechanisms need to be further elucidated.Our studies suggest that the three-lineage differentiation of aged BMSCs can be modulated by culture on hiPSC-engineered ECM. Further studies are aimed at scaling-up to three-dimensional ECM constructs for osteochondral tissue regeneration.