Mesenchymal stromal cell-derived extracellular vesicles for bone regeneration therapy.

PURPOSE: To analyze preclinical bone regeneration studies employing mesenchymal stromal cell (MSC)- derived extracellular vesicles (EVs) and highlight any commonalities in EV biomarker expression, miRNA cargo(s) or pathway activation that will aid in understanding the underlying therapeutic mechanisms. METHODS: Articles employing EVs derived from either MSCs or MSC-like osteogenic stromal cells in preclinical bone regeneration studies are included in this review. RESULTS: EVs derived from a variety of MSC types were able to successfully induce bone formation in preclinical models. Many studies failed to perform in-depth EV characterization. The studies with detailed EV characterization data report very different miRNA cargos, even in EVs isolated from the same species and cell types. Few preclinical studies have analyzed the underlying mechanisms of MSC-EV therapeutic action. CONCLUSION: There is a critical need for mechanistic preclinical studies with thorough EV characterization to determine the best therapeutic MSC-EV source for bone regeneration therapies. Issues including controlled EV delivery, large scale production, and proper storage also need to be addressed before EV-based bone regeneration therapies can be translated for clinical bone repair.

Biomaterial-based extracellular vesicle delivery for therapeutic applications.

Extracellular vesicle (EV)- based therapies have been successfully tested in preclinical models for several biomedical applications, including tissue engineering, drug delivery and cancer therapy. However, EVs are most commonly delivered via local or systemic injection, which results in rapid clearance. In order to prolong the retention of EVs at target site and improve their therapeutic efficacy, biomaterial-based delivery systems are being investigated. This review discusses the various biomaterial-based systems that have been used to deliver EVs for therapeutic applications, specifically highlighting any strategies for controlled release. Further, challenges to clinical translation of biomaterial-based EV delivery systems are also discussed.

The Effects of High-Dose Parathyroid Hormone Treatment on Fusion Outcomes in a Rabbit Model of Posterolateral Lumbar Spinal Fusion Alone and in Combination with Bone Morphogenetic Protein 2 Treatment.

BACKGROUND: Parathyroid hormone (PTH) (1-34) treatment reduces fracture risk in osteoporotic patients. Previously, we demonstrated in a rabbit model that low-dose PTH treatment resulted in increased fusion mass volume. As effects of PTH on bone are dose-dependent, we aimed to evaluate whether increasing dosage of PTH increases both volume and biomechanical stiffness of the resulting fusion masses and/or exhibits synergistic effects with low-dose bone morphogenetic protein 2 (BMP-2). METHODS: Posterolateral lumbar spinal fusion surgery was performed on 60 New Zealand White rabbits divided into 6 experimental groups: iliac crest autograft alone, autograft plus 20 µg/kg/day PTH, autograft plus 40 µg/kg/day PTH, BMP-2 alone, BMP-2 plus 20 µg/kg/day PTH, and BMP-2 plus 40 µg/kg PTH. Fusion was assessed at postoperative week 6 via manual palpation, volumetric computed tomography analysis, and 4-point bending biomechanical testing. RESULTS: All groups treated with BMP-2 fused. Increasing doses of PTH resulted in increased fusion mass volume compared with autograft alone. Autograft plus 40 µg/kg/day PTH yielded fusion mass volumes comparable to BMP-2. When the autograft groups were considered alone, increased mechanical stiffness was observed only in the 20 µg/kg/day group. No significant stiffness differences were observed between BMP-2 groups. CONCLUSIONS: Treatment with the highest dose of PTH resulted in fusion mass volumes similar to those obtained with BMP-2. When the autograft groups were considered alone, significant increases in mechanical stiffness were observed at a dosage of 20 µg/kg/day, suggesting there may be an optimal dose of PTH in the rabbit model. Effects of BMP-2 on fusion were dominant.

Three-Dimensional Printing of Bone Extracellular Matrix for Craniofacial Regeneration.

Tissue-engineered approaches to regenerate bone in the craniomaxillofacial region utilize biomaterial scaffolds to provide structural and biological cues to stem cells to stimulate osteogenic differentiation. Bioactive scaffolds are typically comprised of natural components but often lack the manufacturability of synthetic materials. To circumvent this trade-off, we 3D printed materials comprised of decellularized bone (DCB) matrix particles combined with polycaprolactone (PCL) to create novel hybrid DCB:PCL scaffolds for bone regeneration. Hybrid scaffolds were readily printable at compositions of up to 70% bone by mass and displayed robust mechanical properties. Assessments of surface features revealed both collagenous and mineral components of bone were present. Qualitative and quantitative assessments showed increased surface roughness relative to that of pure PCL scaffolds. These findings correlated with enhanced cell adhesion on hybrid surfaces relative to that on pure surfaces. Human adipose-derived stem cells (hASCs) cultured in DCB:PCL scaffolds without soluble osteogenic cues exhibited significant upregulation of osteogenic genes in hybrid scaffolds relative to pure PCL scaffolds. In the presence of soluble phosphate, hybrid scaffolds resulted in increased calcification. The hASC-seeded scaffolds were implanted into critical-sized murine calvarial defects and yielded greater bone regeneration in DCB:PCL scaffolds compared to that in PCL-only at 1 and 3 months post-transplantation. Taken together, these results demonstrate that 3D printed DCB:PCL scaffolds might be effective for stimulating bone regeneration.

Substrate-mediated gene delivery from glycol-chitosan/hyaluronic acid polyelectrolyte multilayer films.

Substrate-mediated transfection is one of the key strategies for localized gene delivery. Layer-by-layer (LbL) polyelectrolyte deposition is a promising technique which enables controlled delivery of a number of biofactors, including nucleic acids. Here, we embed lipoplexes containing plasmid DNA within polyelectrolyte multilayers composed of glycol-chitosan (Glyc-CHI) and hyaluronic acid (HA) in order to produce a film system that enables localized, surface-based transfection. The topography and morphology of the resulting multilayers were characterized after lipoplex absorption and during subsequent film build-up via atomic force microscopy (AFM) and scanning electron microscopy (SEM), respectively. DNA embedding efficiency and release were then examined. Lipoplex-containing Glyc-CHI/HA films were found to successfully transfect NIH3T3 fibroblasts and HEK293 kidney cells in vitro, maintaining transfection levels of approximately 20% for a period of at least 7 days.

Enhanced MC3T3 preosteoblast viability and adhesion on polyelectrolyte multilayer films composed of glycol-modified chitosan and hyaluronic acid.

Layer-by-layer polyelectrolyte films made of the naturally derived polysaccharides chitosan (CHI) and hyaluronic acid (HA) constitute a well-studied system for the development of cell-responsive biointerfaces. However, many cell lines exhibit decreased adhesion to CHI/HA multilayer films, particularly as the number of bilayers is increased. Here, our group demonstrates that films composed of glycol-modified chitosan exhibit significantly improved MC3T3 preosteoblast adhesion and viability compared to corresponding films consisting of unmodified CHI. These differences in cellular adhesion are likely due to differences in surface topography and roughness, as measured via atomic force microscopy (AFM), as well as in film chemistry and the water solubility of the cation, since both types of films exhibited similar: thickness, as measured via quartz crystal microbalance and AFM; wettability, as measured via contact angle; and serum protein adsorption, as measured via the bicinchoninic acid assay.

Effect of genipin cross-linking on the cellular adhesion properties of layer-by-layer assembled polyelectrolyte films.

Use of polyelectrolyte multi-layers as biomaterials for cell attachment has been limited due to their gel-like characteristics. Herein, we attempt to improve the cellular adhesion properties of multi-layer films, reduce their gel-like nature and rigidify them through chemical cross-linking with genipin; a natural and non-cytotoxic compound. Chitosan (CH), hyaluronan (HA) and alginate (Alg) were used to assemble [CH-HA]n CH and [CH-Alg]n CH films, and the effects of genipin cross-linking on the cell adhesion properties of these multi-layers were investigated. Atomic force microscopy (AFM) confirmed that cross-linking affected each of the films differently. Quartz crystal microbalance with dissipation (QCM-D) revealed that [CH-HA]10 CH films were very viscoelastic, with thicknesses in the range 350-450 nm, while [CH-Alg]10 CH films only grew to thicknesses of approximately 100 nm. These differences were a result of the different growth regimes of these two polyelectrolyte systems. Cell adhesion studies using MC3T3 pre-osteoblasts and rat fibroblastic skin cells, carried out on both films demonstrated vast differences in cell adhesion. [CH-HA]n CH cross-linked films proved to be highly non-adhesive for pre-osteoblasts and fibroblastic skin cells. Conversely, cross-linking [CH-Alg]n CH films was shown to dramatically improve pre-osteoblast and rat fibroblastic skin cell adhesion, especially for high bi-layer numbers and using higher concentrations of cross-linker.