Search

Regeneration of complex bone defects remains a significant clinical challenge. Multi-tool biofabrication has permitted the combination of various biomaterials to create multifaceted composites with tailorable mechanical properties and spatially controlled biological function. In this study we sought to use bioprinting to engineer nonviral gene activated constructs reinforced by polymeric micro-filaments. A gene activated bioink was developed using RGD-γ-irradiated alginate and nano-hydroxyapatite (nHA) complexed to plasmid DNA (pDNA). This ink was combined with bone marrow-derived mesenchymal stem cells (MSCs) and then co-printed with a polycaprolactone supporting mesh to provide mechanical stability to the construct. Reporter genes were first used to demonstrate successful cell transfection using this system, with sustained expression of the transgene detected over 14 days postbioprinting. Delivery of a combination of therapeutic genes encoding for bone morphogenic protein and transforming growth factor promoted robust osteogenesis of encapsulated MSCs in vitro, with enhanced levels of matrix deposition and mineralization observed following the incorporation of therapeutic pDNA. Gene activated MSC-laden constructs were then implanted subcutaneously, directly postfabrication, and were found to support superior levels of vascularization and mineralization compared to cell-free controls. These results validate the use of a gene activated bioink to impart biological functionality to three-dimensional bioprinted constructs.

Photocrosslinkable biomaterials are promising for biomedical applications, as they can be injected in a minimally invasive manner, crosslinked in situ to form hydrogels with cells and/or bioactive factors, and engineered to provide instructive signals to transplanted and host cells. Our group has previously reported on biodegradable, photocrosslinkable alginate (ALG) hydrogels with controlled cell adhesivity for tissue engineering. The polymer backbone of this methacrylated ALG was covalently modified with cell adhesion ligands containing the RGD sequence to enhance the proliferation and differentiation response of encapsulated cells. However, this approach permits limited control over the spatial presentation of these ligands within the three-dimensional hydrogel structure. Here we present a system that easily allows for spatial control of cell adhesion ligands within photocrosslinked ALG hydrogels. A cell adhesive peptide composed of the specific amino acid sequence Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) was covalently modified with acrylate moieties. The acrylated peptide was then covalently incorporated into bulk hydrogels by adding it to methacrylated ALG solutions with a photoinitiator, and then photocrosslinking under long-wave ultraviolet light. The hydrogels were characterized with respect to their swelling and degradation profiles, and the effects of the acrylated peptide on human mesenchymal stem cell (hMSC) viability, adhesion, spreading, and proliferation were examined in vitro. hMSC adhesion and spreading on and proliferation in this biomaterial system could be regulated by varying the concentration of cell adhesion ligand. This new biomaterial system may be a useful platform for tissue engineering, drug delivery, and stem cell transplantation with spatial control of cell adhesivity.

Biocompatible polymers capable of photopolymerization are of immense interest for tissue engineering applications as they can be injected in a minimally invasive manner into a defect site and, then upon application of ultraviolet light, rapidly form hydrogels in situ. Cell adhesion interactions with a biomaterial are known to be important in regulating cell behaviors such as proliferation and differentiation. Therefore, we have covalently modified photocrosslinkable alginate with cell adhesion ligands containing the Arg-Gly-Asp amino acid sequence to form biodegradable, photocrosslinked alginate hydrogels with controlled cell adhesivity. This unique polymer system allows for independent modulation of the physical and biochemical signaling environment presented to cells. The physical properties of the hydrogels such as elastic moduli, swelling ratios, and degradation profiles were similar at the same crosslinking density regardless of the presence of adhesion ligands. Chondrocytes seeded on the surface of the adhesion ligand-modified hydrogels were able to attach and spread, whereas those seeded on unmodified hydrogels exhibited minimal adherence. Importantly, the adhesion-ligand-modified hydrogels enhanced the proliferation and chondrogenic differentiated function of encapsulated chondrocytes as demonstrated by increased DNA content and production of glycosaminoglycans compared to unmodified control hydrogels. This new photocrosslinkable, biodegradable biomaterial system in which the soluble (e.g., growth factors) and insoluble (e.g., cell adhesion ligands) biochemical signaling environment and the biomaterial physical properties (e.g., the elastic moduli) can be independently controlled may be a powerful tool for elucidating the individual and combined effects of these parameters on cell function for cartilage tissue engineering and other regenerative medicine applications.

A number of studies have shown in vivo bone regeneration by transplantation of osteogenic cells differentiated in vitro from adipose-derived stromal cells (ADSCs). However, the in vitro osteogenic differentiation process requires an additional culture period, and the dexamethasone that is generally used in the process may be cytotoxic. Here, we tested the hypothesis that ADSCs that are not differentiated osteogenically in vitro prior to transplantation would extensively regenerate bone in vivo when exogenous bone morphogenetic protein-2 (BMP-2) is delivered to the transplantation site. We fabricated a poly(dl-lactic-co-glycolic acid)/hydroxyapatite (PLGA/HA) composite scaffold with osteoactive HA that is highly exposed on the scaffold surface. This scaffold was able to release BMP-2 over a 4-week period in vitro. Human ADSCs cultured on BMP-2-loaded PLGA/HA scaffolds for 2 weeks differentiated toward osteogenic cells expressing alkaline phosphatase (ALP), osteopontin (OPN), and osteocalcin (OCN) mRNA, while cells on PLGA/HA scaffolds without BMP-2 expressed only ALP. To study in vivo bone formation, PLGA/HA scaffolds (group 1), BMP-2-loaded PLGA/HA scaffolds (group 2), undifferentiated ADSCs seeded on PLGA/HA scaffolds (group 3), and undifferentiated ADSCs seeded on BMP-2-loaded PLGA/HA scaffolds (group 4) were implanted into dorsal, subcutaneous spaces of athymic mice. Eight weeks after implantation, group 4 exhibited a 25-fold greater bone formation area and 5-fold higher calcium deposition than group 3. Bone regeneration by transplanted human ADSCs in group 4 was confirmed by expression of human-specific osteoblastic genes, ALP, collagen type I, OPN, OCN, and bone sialoprotein, while group 3 expressed much lower levels of collagen type I and OPN mRNA only. This study demonstrates the feasibility of extensive in vivo bone regeneration by transplantation of ADSCs without prior in vitro osteogenic differentiation, and that a PLGA/HA composite BMP-2 delivery system stimulates bone regeneration following transplantation of undifferentiated human ADSCs.

Injectable scaffold has raised great interest for tissue regeneration in vivo, because it allows easy filling of irregularly shaped defects and the implantation of cells through minimally invasive surgical procedures. In this study, we evaluated poly(lactic-co-glycolic acid) (PLGA) microsphere as an injectable scaffold for in vivo cartilage tissue engineering. PLGA microspheres (30-80 microm in diameter) were injectable through various gauges of needles, as the microspheres did not obstruct the needles and microsphere size exclusion was not observed at injection. The culture of chondrocytes on PLGA microspheres in vitro showed that the microspheres were permissive for chondrocyte adhesion to the microsphere surface. Rabbit chondrocytes were mixed with PLGA microspheres and injected immediately into athymic mouse subcutaneous sites. Chondrocyte transplantation without PLGA microspheres and PLGA microsphere implantation without chondrocytes served as controls. Four and 9 weeks after implantation, chondrocytes implanted with PLGA microspheres formed solid, white cartilaginous tissues, whereas no gross evidence of cartilage tissue formation was noted in the control groups. Histological analysis of the implants by hematoxylin and eosin staining showed mature and well-formed cartilage. Alcian blue/safranin O staining and Masson's trichrome staining indicated the presence of highly sulfated glycosaminoglycans and collagen, respectively, both of which are the major extracellular matrices of cartilage. Immunohistochemical analysis showed that the collagen was mainly type II, the major collagen type in cartilage. This study demonstrates the feasibility of using PLGA microspheres as an injectable scaffold for in vivo cartilage tissue engineering. This scaffold may be useful to regenerate cartilaginous tissues through minimally invasive surgical procedures in orthopedic, maxillofacial, and urologic applications.