Melanie J. Gupte

Nanofibrous Scaffolds for Dental and Craniofacial Applications

Tissue-engineering solutions often harness biomimetic materials to support cells for functional tissue regeneration. Three-dimensional scaffolds can create a multi-scale environment capable of facilitating cell adhesion, proliferation, and differentiation. One such multi-scale scaffold incorporates nanofibrous features to mimic the extracellular matrix along with a porous network for the regeneration of a variety of tissues. This review will discuss nanofibrous scaffold synthesis/fabrication, biological effects of nanofibers, their tissue- engineering applications in bone, cartilage, enamel, dentin, and periodontium, patient-specific scaffolds, and incorporated growth factor delivery systems. Nanofibrous scaffolds cannot only further the field of craniofacial regeneration but also advance technology for tissue-engineered replacements in many physiological systems.

Development of channeled nanofibrous scaffolds for oriented tissue engineering

A tissue-engineering scaffold resembling the structure of the natural extracellular matrix can often facilitate tissue regeneration. Nerve and tendon are oriented micro-scale tissue bundles. In this study, a method combining injection molding and thermally induced phase separation techniques is developed to create single- and multiple-channeled nanofibrous poly(L-lactic acid) scaffolds. The overall shape, the number and spatial arrangement of channels, the channel wall matrix architecture, the porosity and mechanical properties of the scaffolds are all tunable. The porous NF channel wall matrix provides an excellent microenvironment for protein adsorption and the attachment of PC12 neuronal cells and tendon fibroblast cells, showing potential for neural and tendon tissue regeneration.

Hypoxia differentially regulates human nucleus pulposus and annulus fibrosus cell extracellular matrix production in 3D scaffolds

OBJECTIVE We hypothesize that intervertebral disc (IVD) cells from distinct region respond differently to oxygen environment, and that IVD cells from patients with disc degeneration can benefit from hypoxia condition. Therefore, we aimed to determine the transcriptional response and extracellular matrix (ECM) production of nucleus pulposus (NP) and annulus fibrosus (AF) cells to different oxygen tension. METHOD Human NP and AF from degenerated IVD were seeded in 3D scaffolds and subjected to varying oxygen tension (2% and 20%) for 3 weeks. Changes in ECM were evaluated using quantitative real-time reverse transcriptase polymerase chain reaction, histological and immunohistological analyses. RESULTS Hypoxia significantly enhances NP cells phenotype, which resulted in greater production of sulfated glycosaminoglycan (GAG) and collagen type II within the constructs and the cells expressed higher levels of genes encoding NP ECM. A significantly stronger fluorescent signal for hypoxia-inducible factor (HIF-1α) as also found in the NP cells under the hypoxic than normoxic condition. However, there was little effect of hypoxia on the AF cells. CONCLUSIONS The NP and AF cells respond differently to hypoxia condition on the 3D scaffold, and hypoxia could enhance NP phenotype. When used in concert with appropriate scaffold material, human NP cells from degenerated disc could be regenerated for tissue engineering application.

Biomaterials and stem cells for tissue engineering

Introduction: Organ failure and tissue loss are challenging health issues due to widespread injury, the lack of organs for transplantation and limitations of conventional artificial implants. The field of tissue engineering aims to provide alternative living substitutes that restore, maintain or improve tissue function. Areas covered: In this paper, a wide range of porous scaffolds are reviewed, with an emphasis on phase-separation techniques that generate advantageous nanofibrous 3D scaffolds for stem cell-based tissue engineering applications. In addition, methods for presentation and delivery of bioactive molecules to mimic the properties of stem cell niches are summarized. Recent progress in using these bioinstructive scaffolds to support stem cell differentiation and tissue regeneration is also presented. Expert opinion: Stem cells have great clinical potential because of their capability to differentiate into multiple cell types. Biomaterials have served as artificial extracellular environments to regulate stem cell behavior. Biomaterials with various physical, mechanical and chemical properties can be designed to control stem cell development for regeneration. Conclusion: The research at the interface of stem cell biology and biomaterials has made and will continue to make exciting advances in tissue engineering.

Nanofibrous Spongy Microspheres Enhance Odontogenic Differentiation of Human Dental Pulp Stem Cells

Dentin regeneration is challenging due to its complicated anatomical structure and the shortage of odontoblasts. In this study, a novel injectable cell carrier, nanofibrous spongy microspheres (NF-SMS), is developed for dentin regeneration. Biodegradable and biocompatible poly(l-lactic acid)-block-poly(l-lysine) are synthesized and fabricated into NF-SMS using self-assembly and thermally induced phase separation techniques. It is hypothesized that NF-SMS with interconnected pores and nanofibers can enhance the proliferation and odontogenic differentiation of human dental pulp stem cells (hDPSCs), compared to nanofibrous microspheres (NF-MS) without pore structure and conventional solid microspheres (S-MS) with neither nanofibers nor pore structure. During the first 9 d in culture, hDPSCs proliferate significantly faster on NF-SMS than on NF-MS or S-MS (p < 0.05). Following in vitro odontogenic induction, all the examined odontogenic genes (alkaline phosphatase content, osteocalcin, bone sialoprotein, collagen 1, dentin sialophosphoprotein (DSPP)), calcium content, and DSPP protein content are found significantly higher in the NF-SMS group than in the control groups. Furthermore, 6 weeks after subcutaneous injection of hDPSCs and microspheres into nude mice, histological analysis shows that NF-SMS support superior dentin-like tissue formation compared to NF-MS or S-MS. Taken together, NF-SMS have great potential as an injectable cell carrier for dentin regeneration.

Anterior decompression and nonstructural bone grafting and posterior fixation for cervical facet dislocation with traumatic disc herniation.

Study Design. A series study of patients with lower cervical facet dislocation accompanied by traumatic disc herniation treated with anterior decompression and nonstructural bone grafting and posterior fixation. Objective. To describe a surgical technique of anterior decompression and nonstructural bone grafting and posterior fixation and its clinical outcome in a group of patients with lower cervical facet dislocation accompanied by traumatic disc herniation. Summary of Background Data. The optimal treatment for lower cervical facet dislocation with a prolapsed disc is still controversial. Methods. After discectomy and endplate preparation, a layer of morselized cancellous bone grafts from the iliac crest was placed in the interspace, and held in appropriate sagittal position by 2 layers of gelatin sponge and carefully sutured longus colli muscle. The anterior wound was then closed. The posterior elements were exposed and the reduction was performed. Fluoroscopy was used during reduction maneuver to ensure that the graft was still in the appropriate position. A posterior fusion was performed and the posterior wound was closed. Results. Between January 2006 and February 2010, 21 patients with cervical facet dislocation accompanied by traumatic disc herniation (13 unilateral dislocations and 8 bilateral dislocations) were recruited for this study. All the patients completed at least 1-year follow-up. Average follow-up duration was 29 ± 3.5 months. Average Frankel scales were significantly improved at the end of follow-up, visual analogue scale decreased from 7.8 ± 1.2 before the operation to less than 1.6 ± 0.5 (P < 0.05) 6 months later. Kyposis was corrected from 17.7° ± 6.3° to 6.5° ± 4.1° (P < 0.05) and remained at 5.9° ± 4.2° (P > 0.05) 1 year later. The average subsidence of bone graft was 1.28 ± 0.16 mm at 12 months after the operation and remained 1.34 ± 0.20 mm at 36 months after the operation. All patients had consolidation of both anterior and posterior fusions. No cases of instrument failure occurred and no complications were attributed to the use of this technique. Conclusion. Although further study based on injury types as well as long-term follow-up is still needed, anterior decompression and nonstructural bone grafting and posterior fixation provides a promising surgical option for treating cervical facet dislocation with traumatic disc herniation.

Hypoxia promotes nucleus pulposus phenotype in 3D scaffolds in vitro and in vivo: laboratory investigation.

OBJECT The role of oxygen in disc metabolism remains a matter of debate. Whether the effect of hypoxic priming on the nucleus pulposus phenotype can be maintained in vivo is not clear. The goal of the present study was to test the hypothesis that priming in a low oxygen tension in vitro could promote a nucleus pulposus phenotype in vivo. METHODS Bovine nucleus pulposus cells were seeded in 3D scaffolds and subjected to varying oxygen tensions (2% and 20%) for 3 weeks. The constructs were then implanted subcutaneously for 8 weeks. Changes in the extracellular matrix were evaluated using quantitative real-time reverse transcriptase polymerase chain reaction, glycosaminoglycan (GAG) assay, DNA assay, collagen quantification, and histological and immunohistological analyses. RESULTS Hypoxia resulted in greater production of sulfated glycosaminoglycan and higher levels of gene expression for collagen Type II, aggrecan, and SOX-9. Furthermore, after hypoxic priming, the subcutaneously implanted constructs maintained the nucleus pulposus phenotype, which was indicated by a significantly higher amount of glycosaminoglycan and collagen Type II. CONCLUSIONS Hypoxia enhanced the nucleus pulposus phenotype under experimental conditions both in vitro and in vivo. When used in combination with appropriate scaffold material, nucleus pulposus cells could be regenerated for tissue-engineering applications.

Pore size directs bone marrow stromal cell fate and tissue regeneration in nanofibrous macroporous scaffolds by mediating vascularization

In the U.S., 30% of adults suffer joint pain, most commonly in the knee, which severely limits mobility and is often attributed to injury of cartilage and underlying bone in the joint. Current treatment methods such as microfracture result in less resilient fibrocartilage with eventual failure; autografting can cause donor site morbidity and poor integration. To overcome drawbacks in treatment, tissue engineers can design cell-instructive biomimetic scaffolds using biocompatible materials as alternate therapies for osteochondral defects. Nanofibrous poly (l-lactic acid) (PLLA) scaffolds of uniform, spherical, interconnected and well-defined pore sizes that are fabricated using a thermally-induced phase separation and sugar porogen template method create an extracellular matrix-like environment which facilitates cell adhesion and proliferation. Herein we report that chondrogenesis and endochondral ossification of rabbit and human bone marrow stromal cells (BMSCs) can be controlled by scaffold pore architecture, particularly pore size. Small-pore scaffolds support enhanced chondrogenic differentiation in vitro and cartilage formation in vivo compared to large-pore scaffolds. Endochondral ossification is prevented in scaffolds with very small pore sizes; pore interconnectivity is critical to promote capillary ingrowth for mature bone formation. These results provide a novel strategy to control tissue regenerative processes by tunable architecture of macroporous nanofibrous scaffolds. STATEMENT OF SIGNIFICANCE: Progress in understanding the relationship between cell fate and architectural features of tissue engineering scaffolds is critical for engineering physiologically functional tissues. Sugar porogen template scaffolds have uniform, spherical, highly interconnected macropores. Tunable pore-size guides the fate of bone marrow stromal cells (BMSCs) towards chondrogenesis and endochondral ossification, and is a critical design parameter to mediate neotissue vascularization. Preventing vascularization favors a chondrogenic cell fate while allowing vascularization results in endochondral ossification and mineralized bone formation. These results provide a novel strategy to control tissue regenerative processes by tunable architecture of macroporous nanofibrous scaffolds.