Steven Lu

Osteochondral Tissue Regeneration using a Bilayered Composite Hydrogel with Modulating Dual Growth Factor Release Kinetics in a Rabbit Model

Biodegradable oligo(poly(ethylene glycol) fumarate) (OPF) composite hydrogels have been investigated for the delivery of growth factors (GFs) with the aid of gelatin microparticles (GMPs) and stem cell populations for osteochondral tissue regeneration. In this study, a bilayered OPF composite hydrogel that mimics the distinctive hierarchical structure of native osteochondral tissue was utilized to investigate the effect of transforming growth factor-β3 (TGF-β3) with varying release kinetics and/or insulin-like growth factor-1 (IGF-1) on osteochondral tissue regeneration in a rabbit full-thickness osteochondral defect model. The four groups investigated included (i) a blank control (no GFs), (ii) GMP-loaded IGF-1 alone, (iii) GMP-loaded IGF-1 and gel-loaded TGF-β3, and (iv) GMP-loaded IGF-1 and GMP-loaded TGF-β3 in OPF composite hydrogels. The results of an in vitro release study demonstrated that TGF-β3 release kinetics could be modulated by the GF incorporation method. At 12weeks post-implantation, the quality of tissue repair in both chondral and subchondral layers was analyzed based on quantitative histological scoring. All groups incorporating GFs resulted in a significant improvement in cartilage morphology compared to the control. Single delivery of IGF-1 showed higher scores in subchondral bone morphology as well as chondrocyte and glycosaminoglycan amount in adjacent cartilage tissue when compared to a dual delivery of IGF-1 and TGF-β3, independent of the TGF-β3 release kinetics. The results suggest that although the dual delivery of TGF-β3 and IGF-1 may not synergistically enhance the quality of engineered tissue, the delivery of IGF-1 alone from bilayered composite hydrogels positively affects osteochondral tissue repair and holds promise for osteochondral tissue engineering applications.

Dual growth factor delivery from bilayered, biodegradable hydrogel composites for spatially-guided osteochondral tissue repair

The present work investigated the use of biodegradable hydrogel composite scaffolds, based on the macromer oligo(poly(ethylene glycol) fumarate) (OPF), to deliver growth factors for the repair of osteochondral tissue in a rabbit model. In particular, bilayered OPF composites were used to mimic the structural layers of the osteochondral unit, and insulin-like growth factor-1 (IGF-1) and bone morphogenetic protein-2 (BMP-2) were loaded into gelatin microparticles and embedded within the OPF hydrogel matrix in a spatially controlled manner. Three different scaffold formulations were implanted in a medial femoral condyle osteochondral defect: 1) IGF-1 in the chondral layer, 2) BMP-2 in the subchondral layer, and 3) IGF-1 and BMP-2 in their respective separate layers. The quantity and quality of osteochondral repair was evaluated at 6 and 12 weeks with histological scoring and micro-computed tomography (micro-CT). While histological scoring results at 6 weeks showed no differences between experimental groups, micro-CT analysis revealed that the delivery of BMP-2 alone increased the number of bony trabecular islets formed, an indication of early bone formation, over that of IGF-1 delivery alone. At 12 weeks post-implantation, minimal differences were detected between the three groups for cartilage repair. However, the dual delivery of IGF-1 and BMP-2 had a higher proportion of subchondral bone repair, greater bone growth at the defect margins, and lower bone specific surface than the single delivery of IGF-1. These results suggest that the delivery of BMP-2 enhances subchondral bone formation and that, while the dual delivery of IGF-1 and BMP-2 in separate layers does not improve cartilage repair under the conditions studied, they may synergistically enhance the degree of subchondral bone formation. Overall, bilayered OPF hydrogel composites demonstrate potential as spatially-guided, multiple growth factor release vehicles for osteochondral tissue repair.

Strategies for controlled delivery of biologics for cartilage repair

The delivery of biologics is an important component in the treatment of osteoarthritis and the functional restoration of articular cartilage. Numerous factors have been implicated in the cartilage repair process, but the uncontrolled delivery of these factors may not only reduce their full reparative potential but can also cause unwanted morphological effects. It is therefore imperative to consider the type of biologic to be delivered, the method of delivery, and the temporal as well as spatial presentation of the biologic to achieve the desired effect in cartilage repair. Additionally, the delivery of a single factor may not be sufficient in guiding neo-tissue formation, motivating recent research toward the delivery of multiple factors. This review will discuss the roles of various biologics involved in cartilage repair and the different methods of delivery for appropriate healing responses. A number of spatiotemporal strategies will then be emphasized for the controlled delivery of single and multiple bioactive factors in both in vitro and in vivo cartilage tissue engineering applications.

Injectable dual-gelling cell-laden composite hydrogels for bone tissue engineering

The present work investigated the osteogenic potential of injectable, dual thermally and chemically gelable composite hydrogels for mesenchymal stem cell (MSC) delivery in vitro and in vivo. Composite hydrogels comprising copolymer macromers of N-isopropylacrylamide were fabricated through the incorporation of gelatin microparticles (GMPs) as enzymatically digestible porogens and sites for cellular attachment. High and low polymer content hydrogels with and without GMP loading were shown to successfully encapsulate viable MSCs and maintain their survival over 28 days in vitro. GMP incorporation was also shown to modulate alkaline phosphatase production, but enhanced hydrogel mineralization along with higher polymer content even in the absence of cells. Moreover, the regenerative capacity of 2 mm thick hydrogels with GMPs only, MSCs only, or GMPs and MSCs was evaluated in vivo in an 8 mm rat critical size cranial defect for 4 and 12 weeks. GMP incorporation led to enhanced bony bridging and mineralization within the defect at each timepoint, and direct bone-implant contact as determined by microcomputed tomography and histological scoring, respectively. Encapsulation of both GMPs and MSCs enabled hydrogel degradation leading to significant tissue infiltration and osteoid formation. The results suggest that these injectable, dual-gelling cell-laden composite hydrogels can facilitate bone ingrowth and integration, warranting further investigation for bone tissue engineering.

Evaluation of cell-laden polyelectrolyte hydrogels incorporating poly(l-Lysine) for applications in cartilage tissue engineering

To address the lack of reliable long-term solutions for cartilage injuries, strategies in tissue engineering are beginning to leverage developmental processes to spur tissue regeneration. This study focuses on the use of poly(L-lysine) (PLL), previously shown to up-regulate mesenchymal condensation during developmental skeletogenesis in vitro, as an early chondrogenic stimulant of mesenchymal stem cells (MSCs). We characterized the effect of PLL incorporation on the swelling and degradation of oligo(poly(ethylene) glycol) fumarate) (OPF)-based hydrogels as functions of PLL molecular weight and dosage. Furthermore, we investigated the effect of PLL incorporation on the chondrogenic gene expression of hydrogel-encapsulated MSCs. The incorporation of PLL resulted in early enhancements of type II collagen and aggrecan gene expression and type II/type I collagen expression ratios when compared to blank controls. The presentation of PLL to MSCs encapsulated in OPF hydrogels also enhanced N-cadherin gene expression under certain culture conditions, suggesting that PLL may induce the expression of condensation markers in synthetic hydrogel systems. In summary, PLL can function as an inductive factor that primes the cellular microenvironment for early chondrogenic gene expression but may require additional biochemical factors for the generation of fully functional chondrocytes.

Generation of osteochondral tissue constructs with chondrogenically and osteogenically predifferentiated mesenchymal stem cells encapsulated in bilayered hydrogels

This study investigated the ability of chondrogenic and osteogenic predifferentiation of mesenchymal stem cells (MSCs) to play a role in the development of osteochondral tissue constructs using injectable bilayered oligo(poly(ethylene glycol) fumarate) (OPF) hydrogel composites. We hypothesized that the combinatorial approach of encapsulating cell populations of both chondrogenic and osteogenic lineages in a spatially controlled manner within bilayered constructs would enable these cells to maintain their respective phenotypes via the exchange of biochemical factors even without the influence of external growth factors. During monolayer expansion prior to hydrogel encapsulation, it was found that 7 (CG7) and 14 (CG14) days of MSC exposure to TGF-β3 allowed for the generation of distinct cell populations with corresponding chondrogenic maturities as indicated by increasing aggrecan and type II collagen/type I collagen expression. Chondrogenic and osteogenic cells were then encapsulated within their respective (chondral/subchondral) layers in bilayered hydrogel composites to include four experimental groups. Encapsulated CG7 cells within the chondral layer exhibited enhanced chondrogenic phenotype when compared to other cell populations based on stronger type II collagen and aggrecan gene expression and higher glycosaminoglycan-to-hydroxyproline ratios. Osteogenic cells that were co-cultured with chondrogenic cells (in the chondral layer) showed higher cellularity over time, suggesting that chondrogenic cells stimulated the proliferation of osteogenic cells. Groups with osteogenic cells displayed mineralization in the subchondral layer, confirming the effect of osteogenic predifferentiation. In summary, it was found that MSCs that underwent 7 days, but not 14 days, of chondrogenic predifferentiation most closely resembled the phenotype of native hyaline cartilage when combined with osteogenic cells in a bilayered OPF hydrogel composite, indicating that the duration of chondrogenic preconditioning is an important factor to control. Furthermore, the respective chondrogenic and osteogenic phenotypes were maintained for 28 days in vitro without the need for external growth factors, demonstrating the exciting potential of this novel strategy for the generation of osteochondral tissue constructs for cartilage engineering applications.

Osteochondral Tissue Regeneration Through Polymeric Delivery of DNA Encoding for the SOX Trio and RUNX2

Native osteochondral repair is often inadequate owing to the inherent properties of the tissue, and current clinical repair strategies can result in healing with a limited lifespan and donor site morbidity. This work investigates the use of polymeric gene therapy to address this problem by delivering DNA encoding for transcription factors complexed with the branched poly(ethylenimine)-hyaluronic acid (bPEI-HA) delivery vector via a porous oligo[poly(ethylene glycol) fumarate] hydrogel scaffold. To evaluate the potential of this approach, a bilayered scaffold mimicking native osteochondral tissue organization was loaded with DNA/bPEI-HA complexes. Next, bilayered implants either unloaded or loaded in a spatial fashion with bPEI-HA and DNA encoding for either Runt-related transcription factor 2 (RUNX2) or SRY (sex determining region Y)-box 5, 6, and 9 (the SOX trio), to generate bone and cartilage tissues respectively, were fabricated and implanted in a rat osteochondral defect. At 6weeks post-implantation, micro-computed tomography analysis and histological scoring were performed on the explants to evaluate the quality and quantity of tissue repair in each group. The incorporation of DNA encoding for RUNX2 in the bone layer of these scaffolds significantly increased bone growth. Additionally, a spatially loaded combination of RUNX2 and SOX trio DNA loading significantly improved healing relative to empty hydrogels or either factor alone. Finally, the results of this study suggest that subchondral bone formation is necessary for correct cartilage healing.

Osteochondral defect repair using bilayered hydrogels encapsulating both chondrogenically and osteogenically pre-differentiated mesenchymal stem cells in a rabbit model

OBJECTIVE To investigate the ability of cell-laden bilayered hydrogels encapsulating chondrogenically and osteogenically (OS) pre-differentiated mesenchymal stem cells (MSCs) to effect osteochondral defect repair in a rabbit model. By varying the period of chondrogenic pre-differentiation from 7 (CG7) to 14 days (CG14), the effect of chondrogenic differentiation stage on osteochondral tissue repair was also investigated. METHODS Rabbit MSCs were subjected to either chondrogenic or osteogenic pre-differentiation, encapsulated within respective chondral/subchondral layers of a bilayered hydrogel construct, and then implanted into femoral condyle osteochondral defects. Rabbits were randomized into one of four groups (MSC/MSC, MSC/OS, CG7/OS, and CG14/OS; chondral/subchondral) and received two similar constructs bilaterally. Defects were evaluated after 12 weeks. RESULTS All groups exhibited similar overall neo-tissue filling. The delivery of OS cells when compared to undifferentiated MSCs in the subchondral construct layer resulted in improvements in neo-cartilage thickness and regularity. However, the addition of CG cells in the chondral layer, with OS cells in the subchondral layer, did not augment tissue repair as influenced by the latter when compared to the control. Instead, CG7/OS implants resulted in more irregular neo-tissue surfaces when compared to MSC/OS implants. Notably, the delivery of CG7 cells, when compared to CG14 cells, with OS cells stimulated morphologically superior cartilage repair. However, neither osteogenic nor chondrogenic pre-differentiation affected detectable changes in subchondral tissue repair. CONCLUSIONS Cartilage regeneration in osteochondral defects can be enhanced by MSCs that are chondrogenically and osteogenically pre-differentiated prior to implantation. Longer chondrogenic pre-differentiation periods, however, lead to diminished cartilage repair.

Compositional, structural and mechanical comparisons of normal enamel and hypomaturation enamel.

Hypomaturation amelogenesis imperfecta is a hereditary disorder of the enamel that severely influences the function, aesthetics and psychosocial well-being of patients. In this study, we performed a thorough comparison of normal and hypomaturation enamel through a series of systematical tests on human permanent molars to understand the biomineralization process during pathological amelogenesis. The results of microcomputed tomography, scanning electron microscopy, Fourier transform infrared, Raman spectroscopy, microzone X-ray diffraction, thermal gravimetric analysis, energy diffraction spectrum and Vickers microhardness testing together show dramatic contrasts between hypomaturation enamel and normal enamel in terms of their hierarchical structures, spectral features, crystallographic characteristics, thermodynamic behavior, mineral distribution and mechanical property. Our current study highlights the importance of the organic matrix during the amelogenesis process. It is found that the retention of the organic matrix will influence the quantity, quality and distribution of mineral crystals, which will further demolish the hierarchical architecture of the enamel and affect the related mechanical property. In addition, the high carbonate content in hypomaturation enamel influences the crystallinity, crystal size and solubility of hydroxyapatite crystals. These results deepen our understanding of hypomaturation enamel biomineralization during amelogenesis, explain the clinical manifestations of hypomaturation enamel, provide fundamental evidence to help dentists choose optimal therapeutic strategies and lead to improved biofabrication and gene therapies.

Synthetic biodegradable hydrogel delivery of demineralized bone matrix for bone augmentation in a rat model.

There exists a strong clinical need for a more capable and robust method to achieve bone augmentation, and a system with fine-tuned delivery of demineralized bone matrix (DBM) has the potential to meet that need. As such, the objective of the present study was to investigate a synthetic biodegradable hydrogel for the delivery of DBM for bone augmentation in a rat model. Oligo(poly(ethylene glycol) fumarate) (OPF) constructs were designed and fabricated by varying the content of rat-derived DBM particles (either 1:3, 1:1 or 3:1 DBM:OPF weight ratio on a dry basis) and using two DBM particle size ranges (50-150 or 150-250 μm). The physical properties of the constructs and the bioactivity of the DBM were evaluated. Selected formulations (1:1 and 3:1 with 50-150 μm DBM) were evaluated in vivo compared to an empty control to investigate the effect of DBM dose and construct properties on bone augmentation. Overall, 3:1 constructs with higher DBM content achieved the greatest volume of bone augmentation, exceeding 1:1 constructs and empty implants by 3- and 5-fold, respectively. As such, we have established that a synthetic, biodegradable hydrogel can function as a carrier for DBM, and that the volume of bone augmentation achieved by the constructs correlates directly to the DBM dose.