Browsing by Author "Lam, Johnny"
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Item A factorial analysis of the combined effects of hydrogel fabrication parameters on the in vitro swelling and degradation of oligo(poly(ethylene glycol) fumarate) hydrogels(Wiley, 2014) Lam, Johnny; Kim, Kyobum; Lu, Steven; Tabata, Yasuhiko; Scott, David W.; Mikos, Antonios G.; Kasper, F. KurtisIn this study, a full factorial approach was used to investigate the effects of poly(ethylene glycol) (PEG) molecular weight (MW; 10,000 vs. 35,000 nominal MW), crosslinker-to-macromer carbon–carbon double bond ratio (DBR; 40 vs. 60), crosslinker type (PEG-diacrylate (PEGDA) vs. N,N′-methylene bisacrylamide (MB)), crosslinking extent of incorporated gelatin microparticles (low vs. high), and incubation medium composition (with or without collagenase) on the swelling and degradation characteristics of oligo[(poly(ethylene glycol) fumarate)] (OPF) hydrogel composites as indicated by the swelling ratio and the percentage of mass remaining, respectively. Each factor consisted of two levels, which were selected based on previous in vitro and in vivo studies utilizing these hydrogels for various tissue engineering applications. Fractional factorial analyses of the main effects indicated that the mean swelling ratio and the mean percentage of mass remaining of OPF composite hydrogels were significantly affected by every factor. In particular, increasing the PEG chain MW of OPF macromers significantly increased the mean swelling ratio and decreased the mean percentage of mass remaining by 5.7 ± 0.3 and 17.2 ± 0.6%, respectively. However, changing the crosslinker from MB to PEGDA reduced the mean swelling ratio and increased the mean percentage of mass remaining of OPF composite hydrogels by 4.9 ± 0.2 and 9.4 ± 0.9%, respectively. Additionally, it was found that the swelling characteristics of hydrogels fabricated with higher PEG chain MW or with MB were more sensitive to increases in DBR. Collectively, the main and cross effects observed between factors enables informed tuning of the swelling and degradation properties of OPF-based hydrogels for various tissue engineering applications.Item A rapid, flexible method for incorporating controlled antibiotic release into porous polymethylmethacrylate space maintainers for craniofacial reconstruction(Royal Society of Chemistry, 2016) Mountziaris, Paschalia M.; Shah, Sarita R.; Lam, Johnny; Bennett, George N.; Mikos, Antonios G.Severe injuries in the craniofacial complex, resulting from trauma or pathology, present several challenges to functional and aesthetic reconstruction. The anatomy and position of the craniofacial region make it vulnerable to injury and subsequent local infection due to external bacteria as well as those from neighbouring structures like the sinuses, nasal passages, and mouth. Porous polymethylmethacrylate (PMMA) “space maintainers” have proven useful in staged craniofacial reconstruction by promoting healing of overlying soft tissue prior to reconstruction of craniofacial bones. We describe herein a method by which the porosity of a prefabricated porous PMMA space maintainer, generated by porogen leaching, can be loaded with a thermogelling copolymer-based drug delivery system. Porogen leaching, space maintainer prewetting, and thermogel loading all significantly affected the loading of a model antibiotic, colistin. Weeks-long release of antibiotic at clinically relevant levels was achieved with several formulations. In vitro assays confirmed that the released colistin maintained its antibiotic activity against several bacterial targets. Our results suggest that this method is a valuable tool in the development of novel therapeutic approaches for the treatment of severe complex, infected craniofacial injuries.Item Articular chondrocytes and mesenchymal stem cells seeded on biodegradable scaffolds for the repair of cartilage in a rat osteochondral defect model(Elsevier, 2014) Dahlin, Rebecca L.; Kinard, Lucas A.; Lam, Johnny; Needham, Clark J.; Lu, Steven; Kasper, F. Kurtis; Mikos, Antonios G.This work investigated the ability of co-cultures of articular chondrocytes and mesenchymal stem cells (MSCs) to repair articular cartilage in osteochondral defects. Bovine articular chondrocytes and rat MSCs were seeded in isolation or in co-culture onto electrospun poly(ɛ-caprolactone) (PCL) scaffolds and implanted into an osteochondral defect in the trochlear groove of 12-week old Lewis rats. Additionally, a blank PCL scaffold and untreated defect were investigated. After 12 weeks, the extent of cartilage repair was analyzed through histological analysis, and the extent of bone healing was assessed by quantifying the total volume of mineralized bone in the defect through microcomputed tomography. Histological analysis revealed that the articular chondrocytes and co-cultures led to repair tissue that consisted of more hyaline-like cartilage tissue that was thicker and possessed more intense Safranin O staining. The MSC, blank PCL scaffold, and empty treatment groups generally led to the formation of fibrocartilage repair tissue. Microcomputed tomography revealed that while there was an equivalent amount of mineralized bone formation in the MSC, blank PCL, and empty treatment groups, the defects treated with chondrocytes or co-cultures had negligible mineralized bone formation. Overall, even with a reduced number of chondrocytes, co-cultures led to an equal level of cartilage repair compared to the chondrocyte samples, thus demonstrating the potential for the use of co-cultures of articular chondrocytes and MSCs for the in vivo repair of cartilage defects.Item Autologously Generated Tissue-Engineered Bone Flaps for Reconstruction of Large Mandibular Defects in an Ovine Model(Mary Ann Liebert, Inc., 2015) Tatara, Alexander M.; Kretlow, James D.; Spicer, Patrick P.; Lu, Steven; Lam, Johnny; Liu, Wei; Cao, Yilin; Liu, Guangpeng; Jackson, John D.; Yoo, James J.; Atala, Anthony; van den Beucken, Jeroen J.J.P.; Jansen, John A.; Kasper, F. Kurtis; Ho, Tang; Demian, Nagi; Miller, Michael John; Wong, Mark E.; Mikos, Antonios G.The reconstruction of large craniofacial defects remains a significant clinical challenge. The complex geometry of facial bone and the lack of suitable donor tissue often hinders successful repair. One strategy to address both of these difficulties is the development of an in vivo bioreactor, where a tissue flap of suitable geometry can be orthotopically grown within the same patient requiring reconstruction. Our group has previously designed such an approach using tissue chambers filled with morcellized bone autograft as a scaffold to autologously generate tissue with a predefined geometry. However, this approach still required donor tissue for filling the tissue chamber. With the recent advances in biodegradable synthetic bone graft materials, it may be possible to minimize this donor tissue by replacing it with synthetic ceramic particles. In addition, these flaps have not previously been transferred to a mandibular defect. In this study, we demonstrate the feasibility of transferring an autologously generated tissue-engineered vascularized bone flap to a mandibular defect in an ovine model, using either morcellized autograft or synthetic bone graft as scaffold material.Item Data describing the swelling behavior and cytocompatibility of biodegradable polyelectrolyte hydrogels incorporating poly(L-lysine) for applications in cartilage tissue engineering(Elsevier, 2016) Lam, Johnny; Clark, Elisa C.; Fong, Eliza L.S.; Lee, Esther J.; Lu, Steven; Tabata, Yasuhiko; Mikos, Antonios G.This data article presents data associated with the research article entitled "Evaluation of cell-laden polyelectrolyte hydrogels incorporating poly(L-lysine) for applications in cartilage tissue engineering" (Lam et al., 2016) [1]. Synthetic hydrogel composites fabricated using oligo(poly(ethylene glycol) fumarate) (OPF) macromers were utilized as vehicles for the incorporation of poly(L-lysine) (PLL) as well as the encapsulation of mesenchymal stem cells (MSCs). PLL-laden and PLL-free hydrogels were fabricated to characterize the main and interaction effects of OPF molecular weight, PLL molecular weight, and PLL loading density on the swelling and degradation of synthetic OPF hydrogels. Cells were then encapsulated within such hydrogels for in vitro culture and examined for viability, biochemical activity, and chondrogenic gene expression. These data, which are supplementary to the associated research article (Lam et al., 2016) [1], are presented here.Item Development of Cell-laden Hydrogel Composites for Osteochondral Tissue Engineering(2015-12-14) Lam, Johnny; Mikos, Antonios G.; Grande-Allen, Kathryn J; Zygourakis, Kyriacos; Kasper, Fred KArticular cartilage is a flexible connective tissue that enables the frictionless and painless articulation of bones in synovial joints throughout the body. Given its avascular nature, articular cartilage tissue inherently exhibits a compromised endogenous capacity for regeneration upon damage. Defects caused by disease or trauma often lead to chronic pain and osteoarthritis as current clinical treatments are still unable to achieve long-term repair. Hence, tissue engineers are developing innovative technologies to provide strategies for successful cartilage repair and regeneration. This thesis focuses on the development and evaluation of cell-based osteochondral tissue engineering solutions based on injectable and biodegradable polymer biomaterials for bone and cartilage repair. First, we developed and characterized oligo(poly(ethylene glycol) fumarate) (OPF)-based hydrogels for osteochondral tissue engineering applications. We investigated the main effects of five main hydrogel fabrication parameters (the poly(ethylene glycol) molecular weight (PEG MW), the crosslinker-to-OPF carbon-carbon double bond ratio (DBR), the crosslinker type, the crosslinking density of encapsulated gelatin microparticles, and the incubation medium composition) as well as their interaction effects on the swelling behavior and degradation of OPF hydrogel composites. We found that increasing the PEG MW increased the mean swelling ratio and decreased the mean mass remaining %, while changing the crosslinker type from methylene bisacrylamide (MB) to PEG diacrylate yielded the opposite effect. Additionally, we found that the swelling of hydrogels fabricated with higher PEG MW or with MB were more sensitive to increases in DBR. From these results, we showed that the swelling and degradation properties of OPF-based hydrogels can be precisely tuned through the modulation of these five fabrication parameters. The second part of this thesis investigated the potential of bilayered OPF hydrogel composites encapsulating chondrogenically and osteogenically pre- differentiated MSCs in a spatially controlled fashion for osteochondral tissue repair. We demonstrated that MSCs that underwent 7 days (CG7), but not 14 days (CG14), of chondrogenic pre-differentiation most closely resembled the phenotype of native hyaline cartilage when combined with osteogenically pre-differentiated (OS) cells in a bilayered OPF hydrogel. We found that the respective chondrogenic and osteogenic phenotypes of encapsulated MSCs were maintained for up to 28 days in vitro without the need for external growth factors. When taken in vivo, the delivery of CG7 cells, as opposed to CG14 cells, in combination with OS cells via a bilayered OPF hydrogel composite stimulated morphologically superior cartilage repair. Indeed, the present work showed that 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, resulted in diminished cartilage repair. The final section of this thesis investigated the use of poly(L-lysine) (PLL), previously shown to up-regulate condensation during cartilage development in vitro, as an early chondrogenic stimulant of MSCs encapsulated in OPF hydrogels. We showed that PLL incorporation resulted in early enhancements of type II collagen and aggrecan gene expression as well as increased type II/type I collagen expression ratios when compared to blank controls. We also demonstrated that PLL enhanced N-cadherin gene expression of encapsulated MSCs under certain conditions, suggesting that PLL also likely induced pre-cartilaginous condensation.Item Dual growth factor delivery from bilayered, biodegradable hydrogel composites for spatially-guided osteochondral tissue repair(Elsevier, 2014) Lu, Steven; Lam, Johnny; Trachtenberg, Jordan E.; Lee, Esther J.; Seyednejad, Hajar; van den Beucken, Jeroen J.J.P.; Tabata, Yasuhiko; Wong, Mark E.; Jansen, John A.; Mikos, Antonios G.; Kasper, F. KurtisThe 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.Item Evaluation of cell-laden polyelectrolyte hydrogels incorporating poly(l-Lysine) for applications in cartilage tissue engineering(Elsevier, 2016) Lam, Johnny; Clark, Elisa C.; Fong, Eliza L.S.; Lee, Esther J.; Lu, Steven; Tabata, Yasuhiko; Mikos, Antonios G.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 inᅠvitro, 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.Item Generation of osteochondral tissue constructs with chondrogenically and osteogenically predifferentiated mesenchymal stem cells encapsulated in bilayered hydrogels(Elsevier, 2014) Lam, Johnny; Lu, Steven; Meretoja, Ville V.; Tabata, Yasuhiko; Mikos, Antonios G.; Kasper, F. KurtisThis 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.Item Injectable OPF/graphene oxide hydrogels provide mechanical support and enhance cell electrical signaling after implantation into myocardial infarct(Ivyspring, 2018) Zhou, Jin; Yang, Xiaoning; Liu, Wei; Wang, Chunlan; Shen, Yuan; Zhang, Fengzhi; Zhu, Huimin; Sun, Hongji; Chen, Jiayun; Lam, Johnny; Mikos, Antonios G.; Wang, ChangyongAfter myocardial infarction (MI), the scar tissue contributes to ventricular dysfunction by electrically uncoupling viable cardiomyocytes in the infarct region. Injection of a conductive hydrogel could not only provide mechanical support to the infarcted region, but also synchronize contraction and restore ventricular function by electrically connecting isolated cardiomyocytes to intact tissue. Methods: We created a conductive hydrogel by introducing graphene oxide (GO) nanoparticles into oligo(poly(ethylene glycol) fumarate) (OPF) hydrogels. The hydrogels were characterized by AFM and electrochemistry workstation. A rat model of myocardial infarction was used to investigate the ability of OPF/GO to improve cardiac electrical propagation in the injured heart in vivo. Echocardiography (ECHO) was used to evaluate heart function 4 weeks after MI. Ca2+ imaging was used to visualize beating cardiomyocytes (CMs). Immunofluorescence staining was used to visualize the expression of cardiac-specific markers. Results: OPF/GO hydrogels had semiconductive properties that were lacking in pure OPF. In addition, the incorporation of GO into OPF hydrogels could improve cell attachment in vitro. Injection of OPF/GO 4 weeks after myocardial infarction in rats enhanced the Ca2+ signal conduction of cardiomyocytes in the infarcted region in comparison with PBS or OPF alone. Moreover, the injection of OPF/GO hydrogel into the infarct region enhanced the generation of cytoskeletal structure and intercalated disc assembly. Echocardiography analysis showed improvement in load-dependent ejection fraction/fractional shortening of heart function 4 weeks after injection. Conclusions: We prepared a conductive hydrogel (OPF/GO) that provide mechanical support and biological conduction in vitro and in vivo. We found that injected OPF/GO hydrogels can provide mechanical support and electric connection between healthy myocardium and the cardiomyocytes in the scar via activating the canonical Wnt signal pathway, thus upregulating the generation of Cx43 and gap junction associated proteins. Injection of OPF/GO hydrogel maintained better heart function after myocardial infarction than the injection of a nonconductive polymer.Item Osteochondral tissue regeneration through polymeric delivery of DNA encoding for the SOX trio and RUNX2(Elsevier, 2014) Needham, Clark J.; Shah, Sarita R.; Dahlin, Rebecca L.; Kinard, Lucas A.; Lam, Johnny; Watson, Brendan M.; Lu, Steven; Kasper, F. Kurtis; Mikos, Antonios G.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 6 weeks 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.Item Strategies for controlled delivery of biologics for cartilage repair(Elsevier, 2015) Lam, Johnny; Lu, Steven; Kasper, F. Kurtis; Mikos, Antonios G.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.Item Synthetic biodegradable hydrogel delivery of demineralized bone matrix for bone augmentation in a rat model(Elsevier, 2014) Kinard, Lucas A.; Dahlin, Rebecca L.; Lam, Johnny; Lu, Steven; Lee, Esther J.; Kasper, F. Kurtis; Mikos, Antonios G.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.