A murine model of cutaneous aspergillosis for evaluation of biomaterials-based local delivery therapies.

Cutaneous fungal infection is a challenging condition to treat that primarily afflicts immunocompromised patients. Local antifungal therapy may permit the delivery of high concentrations of antifungals directly to wounds while minimizing systemic toxicities. However, the field currently lacks suitable in vivo models. Therefore, a large cutaneous wound was created in immunosuppressed mice and inoculated with Aspergillus fumigatus. We fabricated biodegradable polymer microparticles (MPs) that were capable of locally delivering antifungal and characterized in vitro release kinetics. We compared wound bed size, fungal burden, and histological presence of fungi in mice treated with antifungal-loaded MPs. Mice with a cutaneous defect but no infection, mice with infected cutaneous defect but no treatment, and infected mice treated with blank MPs were used as controls. Infection of large wounds inhibited healing and resulted in tissue invasion in an inoculum-dependent manner. MPs were capable of releasing antifungals at concentrations above A. fumigatus Minimum Inhibitory Concentration (MIC) for at least 6 days. Wounds treated with MPs had significantly decreased size compared with no treatment (64.2% vs. 19.4% wound reduction, p = 0.002) and were not significantly different from uninfected controls (64.2% vs. 58.1%, p = 0.497). This murine model may serve to better understand cutaneous fungal infection and evaluate local biomaterials-based therapies. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 107A: 1867-1874, 2019.

Data describing the swelling behavior and cytocompatibility of biodegradable polyelectrolyte hydrogels incorporating poly(L-lysine) for applications in cartilage tissue engineering.

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.

In vitro and in vivo evaluation of self-mineralization and biocompatibility of injectable, dual-gelling hydrogels for bone tissue engineering.

In this study, we investigated the mineralization capacity and biocompatibility of injectable, dual-gelling hydrogels in a rat cranial defect as a function of hydrogel hydrophobicity from either the copolymerization of a hydrolyzable lactone ring or the hydrogel polymer content. The hydrogel system comprised a poly(N-isopropylacrylamide)-based thermogelling macromer (TGM) and a polyamidoamine crosslinker. The thermogelling macromer was copolymerized with (TGM/DBA) or without (TGM) a dimethyl-γ-butyrolactone acrylate (DBA)-containing lactone ring that modulated the lower critical solution temperature and thus, the hydrogel hydrophobicity, over time. Three hydrogel groups were examined: (1) 15wt.% TGM, (2) 15wt.% TGM/DBA, and (3) 20wt.% TGM/DBA. The hydrogels were implanted within an 8mm critical size rat cranial defect for 4 and 12weeks. Implants were harvested at each timepoint and analyzed for bone formation, hydrogel mineralization and tissue response using microcomputed tomography (microCT). Histology and fibrous capsule scoring showed a light inflammatory response at 4weeks that was mitigated by 12weeks for all groups. MicroCT scoring and bone volume quantification demonstrated a similar bone formation at 4weeks that was significantly increased for the more hydrophobic hydrogel formulations - 15wt.% TGM and 20wt.% TGM/DBA - from 4weeks to 12weeks. A complementary in vitro acellular mineralization study revealed that the hydrogels exhibited calcium binding properties in the presence of serum-containing media, which was modulated by the hydrogel hydrophobicity. The tailored mineralization capacity of these injectable, dual-gelling hydrogels with hydrolysis-dependent hydrophobicity presents an exciting property for their use in bone tissue engineering applications.

3D tissue-engineered model of Ewing's sarcoma.

Despite longstanding reliance upon monolayer culture for studying cancer cells, and numerous advantages from both a practical and experimental standpoint, a growing body of evidence suggests that more complex three-dimensional (3D) models are necessary to properly mimic many of the critical hallmarks associated with the oncogenesis, maintenance and spread of Ewing's sarcoma (ES), the second most common pediatric bone tumor. And as clinicians increasingly turn to biologically-targeted therapies that exert their effects not only on the tumor cells themselves, but also on the surrounding extracellular matrix, it is especially important that preclinical models evolve in parallel to reliably measure antineoplastic effects and possible mechanisms of de novo and acquired drug resistance. Herein, we highlight a number of innovative methods used to fabricate biomimetic ES tumors, encompassing both the surrounding cellular milieu and the extracellular matrix (ECM), and suggest potential applications to advance our understanding of ES biology, preclinical drug testing, and personalized medicine.

Hydrogel-based 3D model of patient-derived prostate xenograft tumors suitable for drug screening.

The lack of effective therapies for bone metastatic prostate cancer (PCa) underscores the need for accurate models of the disease to enable the discovery of new therapeutic targets and to test drug sensitivities of individual tumors. To this end, the patient-derived xenograft (PDX) PCa model using immunocompromised mice was established to model the disease with greater fidelity than is possible with currently employed cell lines grown on tissue culture plastic. However, poorly adherent PDX tumor cells exhibit low viability in standard culture, making it difficult to manipulate these cells for subsequent controlled mechanistic studies. To overcome this challenge, we encapsulated PDX tumor cells within a three-dimensional hyaluronan-based hydrogel and demonstrated that the hydrogel maintains PDX cell viability with continued native androgen receptor expression. Furthermore, a differential sensitivity to docetaxel, a chemotherapeutic drug, was observed as compared to a traditional PCa cell line. These findings underscore the potential impact of this novel 3D PDX PCa model as a diagnostic platform for rapid drug evaluation and ultimately push personalized medicine toward clinical reality.

Phosphorous-containing polymers for regenerative medicine.

Disease and injury have resulted in a large, unmet need for functional tissue replacements. Polymeric scaffolds can be used to deliver cells and bioactive signals to address this need for regenerating damaged tissue. Phosphorous-containing polymers have been implemented to improve and accelerate the formation of native tissue both by mimicking the native role of phosphorous groups in the body and by attachment of other bioactive molecules. This manuscript reviews the synthesis, properties, and performance of phosphorous-containing polymers that can be useful in regenerative medicine applications.

Evaluation of antibiotic releasing porous polymethylmethacrylate space maintainers in an infected composite tissue defect model.

This study evaluated the in vitro and in vivo performance of antibiotic-releasing porous polymethylmethacrylate (PMMA)-based space maintainers comprising a gelatin hydrogel porogen and a poly(dl-lactic-co-glycolic acid) (PLGA) particulate carrier for antibiotic delivery. Colistin was released in vitro from either gelatin or PLGA microparticle loaded PMMA constructs, with gelatin-loaded constructs releasing colistin over approximately 7 days and PLGA microparticle-loaded constructs releasing colistin for up to 8 weeks. Three formulations with either burst release or extended release at different doses were tested in a rabbit mandibular defect inoculated with Acinetobacter baumannii (2×10(7) colony forming units ml(-1)). In addition, one material control that released antibiotic but was not inoculated with A. baumannii was tested. A. baumannii was not detectable in any animal after 12 weeks on culture of the defect, saliva, or blood. Defects with high dose extended release implants had greater soft tissue healing compared with defects with burst release implants, with 8 of 10 animals showing healed mucosae compared with 2 of 10 respectively. Extended release of locally delivered colistin via a PLGA microparticle carrier improved soft tissue healing compared with implants with burst release of colistin from a gelatin carrier.

Effect of temporally patterned TNF-α delivery on in vitro osteogenic differentiation of mesenchymal stem cells cultured on biodegradable polymer scaffolds.

Recent insight into the critical role of pro-inflammatory cytokines, particularly tumor necrosis factor-α (TNF-α), in bone regeneration has heralded a new direction in the design of tissue engineering constructs. Previous studies have demonstrated that continuous delivery of 50 ng/ml TNF-α to mesenchymal stem cells (MSCs) cultured on three-dimensional (3D) biodegradable electrospun poly(ϵ-caprolactone) (PCL) microfiber meshes stimulates mineralized matrix deposition, a marker of osteogenic differentiation. Since TNF-α exhibits a biphasic pattern of expression following bone fracture in vivo, this study aimed to investigate the effects of temporal patterns of TNF-α delivery on in vitro osteogenic differentiation of MSCs cultured on 3D electrospun PCL scaffolds. MSCs were cultured for 16 days and exposed to continuous, early, intermediate, or late TNF-α delivery. To further elucidate the effects of TNF-α on osteogenic differentiation, the study design included MSCs precultured both in the presence and absence of typically required osteogenic supplement dexamethasone. Mineralized matrix deposition was not observed in constructs with dexamethasone-naïve MSCs, suggesting that TNF-α is not sufficient to trigger in vitro osteogenic differentiation of MSCs. For MSCs precultured with dexamethasone, TNF-α suppressed alkaline phosphatase activity, an early marker of osteogenic differentiation, and stimulated mineralized matrix deposition, a late stage marker of MSC osteogenic differentiation. By elucidating the impact of temporal variations in TNF-α delivery on MSC osteogenic differentiation, our results offer insight into the regenerative mechanism of TNF-α and provide the design parameters for a novel tissue engineering strategy that rationally controls TNF-α signaling to stimulate bone regeneration.

Student Award for Outstanding Research Winner in the Ph.D. Category for the 9th World Biomaterials Congress, Chengdu, China, June 1-5, 2012: The interplay of bone-like extracellular matrix and TNF-α signaling on in vitro osteogenic differentiation of mesenchymal stem cells.

As an initial step in the development of a bone tissue engineering strategy to rationally control inflammation, we investigated the interplay of bone-like extracellular matrix (ECM) and varying doses of the inflammatory cytokine tumor necrosis factor alpha (TNF-α) on osteogenically differentiating mesenchymal stem cells (MSCs) cultured in vitro on 3D poly(ε-caprolactone) (PCL) microfiber scaffolds containing pregenerated bone-like ECM. To generate the ECM, PCL scaffolds were seeded with MSCs and cultured in medium containing the typically required osteogenic supplement dexamethasone. However, since dexamethasone antagonizes TNF-α, the interplay of ECM and TNF-α was investigated by culturing naïve MSCs on the decellularized scaffolds in the absence of dexamethasone. MSCs cultured on ECM-coated scaffolds continued to deposit mineralized matrix, a late stage marker of osteogenic differentiation. Mineralized matrix deposition was not adversely affected by exposure to TNF-α for 4-8 days, but was significantly reduced after continuous exposure to TNF-α over 16 days, which simulates the in vivo response, where brief TNF-α signaling stimulates bone regeneration, while prolonged exposure has damaging effects. This underscores the exciting potential of PCL/ECM constructs as a more clinically realistic in vitro culture model to facilitate the design of new bone tissue engineering strategies that rationally control inflammation to promote regeneration.

Chondrogenic differentiation of neonatal human dermal fibroblasts encapsulated in alginate beads with hydrostatic compression under hypoxic conditions in the presence of bone morphogenetic protein-2.

In the present work, neonatal human dermal fibroblasts (nHDFs) were evaluated as a potential cell source for intervertebral disc repair. Chondrogenic differentiation of nHDFs was studied in the presence or absence of hydrostatic compression under normal and hypoxic conditions. In addition, the potentially synergistic effects of mechanical stimulation and bone morphogenetic protein (BMP)-2 on the chondrogenic differentiation of nHDFs were assessed. Mechanical stimulation was applied to the cells encapsulated in alginate beads using a custom-built bioreactor system for either a 1- or 3-week period at a frequency of 1 Hz for 4 h/day. In general, after 21 days of culture, high cell viability was observed for all the groups, with the exception of the groups exposed to intermittent mechanical stimulation for 3 weeks. Long-term intermittent application of mechanical stimulation under low O(2) conditions resulted in elevated collagen biosynthesis rate from day 0. Inclusion of BMP-2 for this group improved the chondrogenic differentiation of nHDFs, as indicated by elevated aggrecan gene expression and an increased collagen production rate compared to the day 0 group. Thus, the combination of hypoxia, BMP-2 supplementation, and long-term intermittent application of dynamic hydrostatic pressure was found to be a potent promoter of the chondrogenic differentiation of nHDFs.

Investigating the role of hematopoietic stem and progenitor cells in regulating the osteogenic differentiation of mesenchymal stem cells in vitro.

Significant progress has been made in understanding the hematopoietic supportive capacity of both mesenchymal stem cells (MSCs) and osteogenic cells in maintaining hematopoietic stem and progenitor cells (HSPCs) in vitro. However the role of HSPCs in regulating their bone marrow niche environment through influencing the function of neighboring cell populations to complete this reciprocal relationship is not well understood. In this study, we investigated the influence of HSPCs on the osteogenic differentiation of MSCs in vitro, using a highly enriched population of hematopoietic cells with the phenotype c-Kit(+)Sca-1(+)Lineage(-)(KSL) and bone marrow derived mesenchymal stromal cells in direct contact co-culture in medium with or without the addition of the osteogenic supplement dexamethasone. The data suggest that a low dose of HSPCs in co-culture with MSCs in combination with dexamethasone treatment accelerates the osteogenic progression of MSCs, as evidenced in the earlier peak in alkaline phosphatase activity and enhanced calcium deposition compared to cultures of MSCs alone. We observed a longer persistence of functional primitive hematopoietic stem and progenitor cells in the population treated with dexamethasone, and this observation was positively correlated with enhanced osteogenic differentiation of MSCs. Therefore, our findings further support the concept that HSPCs are actively involved in regulating the development and maintenance of the stem cell niche environment in which they reside.

Thermoresponsive, in situ cross-linkable hydrogels based on N-isopropylacrylamide: fabrication, characterization and mesenchymal stem cell encapsulation.

Hydrogels that solidify in response to a dual, physical and chemical, mechanism upon temperature increase were fabricated and characterized. The hydrogels were based on N-isopropylacrylamide, which renders them thermoresponsive, and contained covalently cross-linkable moieties in the macromers. The effects of the macromer end group, acrylate or methacrylate, and the fabrication conditions on the degradative and swelling properties of the hydrogels were investigated. The hydrogels exhibited higher swelling below their lower critical solution temperature (LCST). When immersed in cell culture medium at physiological temperature, which was above their LCST, hydrogels showed constant swelling and no degradation over 8 weeks, with the methacrylated hydrogels showing greater swelling than their acrylated analogs. In addition, hydrogels immersed in cell culture medium under the same conditions showed lower swelling compared with phosphate-buffered saline. The interplay between chemical cross-linking and thermally induced phase separation affected the swelling characteristics of the hydrogels in different media. Mesenchymal stem cells encapsulated in the hydrogels in vitro were viable over 3 weeks and markers of osteogenic differentiation were detected when the cells were cultured with osteogenic supplements. Hydrogel mineralization in the absence of cells was observed in cell culture medium with the addition of fetal bovine serum and ß-glycerol phosphate. The results suggest that these hydrogels may be suitable as carriers for cell delivery in tissue engineering.

Combination of enzymes and flow perfusion conditions improves osteogenic differentiation of bone marrow stromal cells cultured upon starch/poly(epsilon-caprolactone) fiber meshes.

Previous studies have shown that alpha-amylase and lipase are capable of enhancing the degradation of fiber meshes blends of starch and poly(epsilon-caprolactone) (SPCL) under dynamic conditions, and consequently to promote the proliferation and osteogenic differentiation of bone marrow stromal cells (MSCs). This study investigated the effect of flow perfusion bioreactor culture in combination with enzymes on the osteogenic differentiation of MSCs. SPCL fiber meshes were seeded with MSCs and cultured with osteogenic medium supplemented with alpha-amylase, lipase, or a combination of the two for 8 or 16 days using static or flow conditions. Lipase and its combination with alpha-amylase enhanced cell proliferation after 16 days. In addition, the flow perfusion culture enhanced the infiltration of cells and facilitated greater distribution of extracellular matrix (ECM) throughout the scaffolds in the presence/absence of enzymes. A significant amount of calcium was detected after 16 days in all groups cultured in flow conditions compared with static cultures. Nevertheless, when alpha-amylase and lipase were included in the flow perfusion cultures, the calcium content was 379 +/- 30 microg/scaffold after as few as 8 days. The highest calcium content (1271 +/- 32 microg/scaffold) was obtained for SPCL/cell constructs cultured for 16 days in the presence of lipase and flow. Furthermore, von Kossa staining and tetracycline fluorescence of histological sections demonstrated mineral deposition within the scaffolds for all groups cultured for 16 days under flow. However, all the data corroborate that lipase coupled with flow perfusion conditions improve the osteogenic differentiation of MSCs and enhance ECM mineralization.

Engineering tumors: a tissue engineering perspective in cancer biology.

There is increasing recognition that three-dimensional (3D) tissue culture technologies have many uses within the biomedical sciences beyond the scope of regenerative medicine. One such use is in the field of cancer biology, where a 3D tumor model that accurately recreates the in vivo tumor phenotype would be a valuable tool for studying tumor biology and would allow better preclinical evaluation of anticancer drug candidates. The most widely used model involves small cellular aggregates, termed spheroids, which have been used by cancer biologists for decades and have consistently shown the superiority of 3D tissue culture over standard two-dimensional monolayer culture for mimicking the tumor behavior and drug resistance encountered in vivo. Currently, several research groups have begun to adapt more advanced 3D culture techniques from the tissue engineering field to create a more clinically accurate ex vivo model of tumor biology.

Modulation of osteogenic properties of biodegradable polymer/extracellular matrix scaffolds generated with a flow perfusion bioreactor.

In this study, composite scaffolds consisting of both synthetic and natural components with controllable properties were generated by incorporating mineralized extracellular matrix (ECM) and electrospun poly(epsilon-caprolactone) (PCL) microfiber scaffolds. Mesenchymal stem cells (MSCs) were cultured on PCL scaffolds under flow perfusion conditions with culture medium supplemented with dexamethasone to investigate the effect of culture duration on mineralized extracellular matrix deposition. MSCs differentiated down the osteogenic lineage and produced extracellular matrix with different compositions of mineral, collagen, and glycosaminoglycan with distinct morphologies at various stages of osteogenesis. To determine whether the presence and maturity of mineralized extracellular matrix influences osteogenic differentiation in vitro, PCL/ECM constructs were decellularized to yield PCL/ECM composite scaffolds that were subsequently seeded with MSCs and cultured in the absence of dexamethasone. The presence of mineralized matrix reduced cellular proliferation while stimulating alkaline phosphatase activity with increasing amounts of calcium deposition over time. PCL/ECM composite scaffolds containing the most mature mineralized matrix resulted in the most rapid increase and highest levels of alkaline phosphatase activity and calcium deposition compared to all other scaffold groups. Therefore, we demonstrate that mineralized extracellular matrix generated under controlled flow perfusion conditions can impart osteogenic properties to an osteoconductive polymer scaffold, and that the maturity of this matrix influences osteogenic differentiation in vitro, even in the absence of dexamethasone.

Osteogenic differentiation of mesenchymal stem cells on pregenerated extracellular matrix scaffolds in the absence of osteogenic cell culture supplements.

This study utilized a full-factorial design to investigate the effect of four factors: presence of whole bone marrow cells, presence of in vitro-generated mineralized extracellular matrix (ECM), presence of dexamethasone, and variations in culture duration, on the proliferation and osteogenic differentiation of mesenchymal stem cells (MSCs) cultured on a polymer scaffold. Electrospun poly(epsilon-caprolactone) (PCL) fiber mesh scaffolds were seeded with rat MSCs and cultured in complete osteogenic medium for 12 days to generate constructs containing mineralized ECM. MSCs or MSCs and whole bone marrow cells were seeded onto decellularized ECM constructs (PCL/ECM) or plain PCL scaffolds and cultured statically for 4, 8, and 16 days in medium either with or without dexamethasone. After each culture period, the cell number was determined by DNA analysis, and the osteogenic differentiation state of the cells was determined by alkaline phosphatase activity and calcium assays. MSCs seeded onto PCL/ECM constructs and cultured in medium either with or without dexamethasone demonstrated similar amounts of calcium deposition after 16 days. A significant increase in cell number over time compared with all other groups was observed when whole bone marrow cells were cocultured with MSCs on PCL scaffolds in medium without dexamethasone. This study establishes that the osteogenic differentiation of MSCs seeded onto ECM-containing constructs is maintained even in the absence of dexamethasone and that the coculture of MSCs and whole bone marrow cells without dexamethasone and ECM enhances the proliferation of a cell population (or populations) present in the whole bone marrow.

In vivo bone biocompatibility and degradation of porous fumarate-based polymer/alumoxane nanocomposites for bone tissue engineering.

The objective of this study was to determine how the incorporation of surface-modified alumoxane nanoparticles into a biodegradable fumarate-based polymer affects in vivo bone biocompatibility (characterized by direct bone contact and bone ingrowth) and in vivo degradability. Porous scaffolds were fabricated from four materials: poly(propylene fumarate)/propylene fumarate-diacrylate (PPF/PF-DA) polymer alone; a macrocomposite consisting of PPF/PF-DA polymer with boehmite microparticles; a nanocomposite composed of PPF/PF-DA polymer and mechanically reinforcing surface-modified alumoxane nanoparticles; and a low-molecular weight PPF polymer alone (tested as a degradation control). Scaffolds were implanted in the lateral femoral condyle of adult goats for 12 weeks and evaluated by micro-computed tomography and histological analysis. For all material groups, small amounts of bone, some soft tissue, and a few inflammatory elements were observed within the pores of scaffolds, though many pores remained empty or filled with fluid only. Direct contact between scaffolds and surrounding bone tissue was also observed in all scaffold types, though less commonly. Minimal in vivo degradation occurred during the 12 weeks of implantation in all materials except the degradation control. These results demonstrate that the incorporation of alumoxane nanoparticles into porous PPF/PF-DA scaffolds does not significantly alter in vivo bone biocompatibility or degradation.

Dose effect of tumor necrosis factor-alpha on in vitro osteogenic differentiation of mesenchymal stem cells on biodegradable polymeric microfiber scaffolds.

This study presents a first step in the development of a bone tissue engineering strategy to trigger enhanced osteogenesis by modulating inflammation. This work focused on characterizing the effects of the concentration of a pro-inflammatory cytokine, tumor necrosis factor alpha (TNF-alpha), on osteogenic differentiation of mesenchymal stem cells (MSCs) grown in a 3D culture system. MSC osteogenic differentiation is typically achieved in vitro through a combination of osteogenic supplements that include the anti-inflammatory corticosteroid dexamethasone. Although simple, the use of dexamethasone is not clinically realistic, and also hampers in vitro studies of the role of inflammatory mediators in wound healing. In this study, MSCs were pre-treated with dexamethasone to induce osteogenic differentiation, and then cultured in biodegradable electrospun poly(epsilon-caprolactone) (PCL) scaffolds, which supported continued MSC osteogenic differentiation in the absence of dexamethasone. Continuous delivery of 0.1 ng/mL of recombinant rat TNF-alpha suppressed osteogenic differentiation of rat MSCs over 16 days, which was likely the result of residual dexamethasone antagonizing TNF-alpha signaling. Continuous delivery of a higher dose, 5 ng/mL TNF-alpha, stimulated osteogenic differentiation for a few days, and 50 ng/mL TNF-alpha resulted in significant mineralized matrix deposition over the course of the study. These findings suggest that the pro-inflammatory cytokine TNF-alpha stimulates osteogenic differentiation of MSCs, an effect that can be blocked by the presence of anti-inflammatory agents like dexamethasone, with significant implications on the interplay between inflammation and tissue regeneration.

The role of lipase and alpha-amylase in the degradation of starch/poly(epsilon-caprolactone) fiber meshes and the osteogenic differentiation of cultured marrow stromal cells.

The present work studies the influence of hydrolytic enzymes (alpha-amylase or lipase) on the degradation of fiber mesh scaffolds based on a blend of starch and poly(epsilon-caprolactone) (SPCL) and the osteogenic differentiation of osteogenic medium-expanded rat bone marrow stromal cells (MSCs) and subsequent formation of extracellular matrix on these scaffolds under static culture conditions. The biodegradation profile of SPCL fiber meshes was investigated using enzymes that are specifically responsible for the enzymatic hydrolysis of SPCL using concentrations similar to those found in human serum. These degradation studies were performed under static and dynamic conditions. After several degradation periods (3, 7, 14, 21, and 30 days), weight loss measurements and micro-computed tomography analysis (specifically porosity, interconnectivity, mean pore size, and fiber thickness) were performed. The SPCL scaffolds were seeded with rat MSCs and cultured for 8 and 16 days using complete osteogenic media with and without enzymes (alpha-amylase or lipase). Results indicate that culture medium supplemented with enzymes enhanced cell proliferation after 16 days of culture, whereas culture medium without enzymes did not. No calcium was detected in groups cultured with alpha-amylase or without enzymes after each time period, although groups cultured with lipase presented calcium deposition after the eighth day, showing a significant increase at the sixteenth day. Lipase appears to positively influence osteoblastic differentiation of rat MSCs and to enhance matrix mineralization. Furthermore, scanning electron microscopy images showed that the enzymes did not have a deleterious effect on the three-dimensional structure of SPCL fiber meshes, meaning that the scaffolds did not lose their structural integrity after 16 days. Confocal micrographs have shown cells to be evenly distributed and infiltrated within the SPCL fiber meshes up to 410 microm from the surface. This study demonstrates that supplementation of culture media with lipase holds great potential for the generation of bone tissue engineering constructs from MSCs seeded onto SPCL fiber meshes, because lipase enhances the osteoblastic differentiation of the seeded MSCs and promotes matrix mineralization without harming the structural integrity of the meshes over 16 days of culture.

Fabrication and in vitro degradation of porous fumarate-based polymer/alumoxane nanocomposite scaffolds for bone tissue engineering.

In this work, the fabrication and in vitro degradation of porous fumarate-based/alumoxane nanocomposites were evaluated for their potential as bone tissue engineering scaffolds. The biodegradable polymer poly (propylene fumarate)/propylene fumarate-diacrylate (PPF/PF-DA), a macrocomposite composed of PPF/PF-DA and boehmite microparticles, and a nanocomposite composed of PPF/PF-DA and surface-modified alumoxane nanoparticles were used to fabricate porous scaffolds by photo-crosslinking and salt-leaching. Scaffolds then underwent 12 weeks of in vitro degradation in phosphate buffered saline at 37 degrees C. The presence of boehmite microparticles and alumoxane nanoparticles in the polymer inhibited scaffold shrinkage during crosslinking. Furthermore, the incorporation of alumoxane nanoparticles into the polymer limited salt-leaching, perhaps due to tighter crosslinking within the nanocomposite. Analysis of crosslinking revealed that the acrylate and overall double bond conversions in the nanocomposite were higher than in the PPF/PF-DA polymer alone, though these differences were not significant. During 12 weeks of in vitro degradation, the nanocomposite lost 5.3% +/- 2.4% of its mass but maintained its compressive mechanical properties and porous architecture. The addition of alumoxane nanoparticles into the fumarate-based polymer did not significantly affect the degradation of the nanocomposite compared with the other materials in terms of mass loss, compressive properties, and porous structure. These results demonstrate the feasibility of fabricating degradable nanocomposite scaffolds for bone tissue engineering by photo-crosslinking and salt-leaching mixtures of fumarate-based polymers, alumoxane nanoparticles, and salt microparticles.