Yang-Jo Seol

Enhanced bone formation by controlled growth factor delivery from chitosan-based biomaterials.

For the purpose of obtaining high bone forming efficacy, development of chitosan was attempted as a tool useful as a scaffolding device. Porous chitosan matrices, chitosan-poly(L-lactide) (PLLA) composite matrices and chitosan coated on PLLA matrices were dealt with in this research. Porous chitosan matrix was fabricated by freeze-drying and cross-linking aqueous chitosan solution. Porous chitosan matrix combined with ceramics and constituents of extracellular matrices were prepared and examined for their bone regenerative potential. Composite porous matrix of chitosan-PLLA was prepared by mixing polylactide with chitosan and freeze-drying. All chitosan based devices demonstrated improved bone forming capacity by increasing mechanical stability and biocompatibility. Release of platelet-derived growth factor-BB (PDGF-BB) from these matrices exerted significant osteoinductive effect in addition to the high osteoconducting capacity of the porous chitosan matrices. The hydrophobic surface of PLLA matrices was modified by chitosan to enhance cell affinity and wettability. The chitosan coated PLLA matrix induced increased osteoblast attachment as compared with intact PLLA surface. Overall results in this study demonstrated the usefulness of chitosan as drug releasing scaffolds and as modification tools for currently used biomaterials to enhance tissue regeneration efficacy. These results may expand the feasibility of combinative strategy of controlled local drug delivery concept and tissue engineered bone formation in reconstructive therapy in the field of periodontics, orthopedics and plastic surgery.

Controlled release of platelet-derived growth factor-BB from chondroitin sulfate-chitosan sponge for guided bone regeneration.

Platelet-derived growth factor-BB (PDGF-BB) releasing porous chondroitin-4-sulfate (CS)-chitosan sponge was designed with an aim of controlling growth factor delivery in order to improve bone formation. Porous CS-chitosan sponge was fabricated by freeze drying and crosslinking aqueous CS-chitosan solution. PDGF-BB was incorporated into the CS-chitosan sponge by soaking CS-chitosan sponge into the PDGF-BB solution. CS-chitosan sponge retained a porous structure with a 150-200-microm pore diameter that was suitable for cellular migration and osteoid ingrowth. Release rate of PDGF-BB could be controlled by varying the composition of CS in the sponge or initial loading content of PDGF-BB. CS-chitosan sponge induced increased osteoblast migration and proliferation as compared with chitosan sponge alone. Furthermore, the release of PDGF-BB from CS-chitosan sponge significantly enhanced osteoblast proliferation. These results suggest that PDGF-BB-releasing CS-chitosan sponge may be beneficial to enhance bone cell adaptation and regenerative potential when applied in wound sites.

Alveolar Bone Regeneration by Transplantation of Periodontal Ligament Stem Cells and Bone Marrow Stem Cells in a Canine Peri-Implant Defect Model: A Pilot Study

BACKGROUND The present study was undertaken to evaluate the potential of periodontal ligament stem cells (PDLSCs) and bone marrow SCs (BMSCs) on alveolar bone regeneration in a canine peri-implant defect model. METHODS Four adult, male beagle dogs were used in this study. Autologous BMSCs from the iliac crests and PDLSCs from extracted teeth were cultured. Three months after extraction, BMSC- and PDLSC-loaded hydroxyapatite/beta-tricalcium phosphate (HA/TCP) (test groups) and cell-free HA/TCP (control group) were implanted in three rectangular, saddle-like peri-implant defects, respectively. The left side of the mandible was initially prepared, and after 8 weeks, the right side was also prepared. The animals were sacrificed after an 8-week healing period. Undecalcified ground sections were prepared. New bone formation and bone-to-implant contact (BIC) were measured histomorphometrically. BMSCs and PDLSCs were fluorescently labeled and traced. RESULTS Alveolar bone regeneration in surgically created peri-implant saddle-like defects was more effective in test groups than the control group. The BMSC group had the highest new bone formation (34.99% and 40.17% at healing times of 8 and 16 weeks, respectively) followed by the PDLSC group (31.90% and 36.51%) and control group (23.13% and 28.36%), respectively. Test groups exhibited a significantly higher new bone formation than the control group at 8 weeks, but the same was true for only the BMSC group at 16 weeks (P <0.05). Fluorescently labeled cells were identified adjacent to HA/TCP carriers and, partly, near connective tissues and osteoids. CONCLUSION This study demonstrated the feasibility of using stem cell-mediated bone regeneration to treat peri-implant defects.

Tissue-engineered growth of bone by marrow cell transplantation using porous calcium metaphosphate matrices

In this study we investigated not only osteoblastic cell proliferation and differentiation on the surface of calcium metaphosphate (CMP) matrices in vitro but also bone formation by ectopic implantation of these cell-matrix constructs in athymic mice in vivo. Interconnected porous CMP matrices with pores 200 microm in size were prepared to use as scaffolds for rat-marrow stromal-cell attachment. Cell-matrix constructs were cultured in vitro, and cell proliferation and ALPase activities were monitored for 56 days. In addition to their being cultured in vitro, cell-matrix constructs were implanted into subcutaneous sites of athymic mice. In vitro these porous CMP matrices supported the proliferation of osteoblastic cells as well as their differentiation, as indicated by high ALPase activity. In vivo the transplanted marrow cells gave rise to bone tissues in the pores of the CMP matrices. A small amount of woven bone formation was detected first at 4 weeks; osteogenesis progressed vigorously with time, and thick lamellar bones that had been remodeled were observed at 12 weeks. These findings demonstrate the potential for using a porous CMP matrix as a biodegradable scaffold ex vivo along with attached marrow-derived mesenchymal cells for transplantation into a site for bone regeneration in vivo.

Pre-clinical Models for Oral and Periodontal Reconstructive Therapies

The development of new medical formulations (NMF) for reconstructive therapies has considerably improved the available treatment options for individuals requiring periodontal repair or oral implant rehabilitation. Progress in tissue engineering and regenerative medicine modalities strongly depends on validated pre-clinical research. Pre-clinical testing has contributed to the recent approval of NMF such as GEM 21S® and INFUSE® bone grafts for periodontal and oral regenerative therapies. However, the selection of a suitable pre-clinical model for evaluation of the safety and efficacy of a NMF remains a challenge. This review is designed to serve as a primer to choose the appropriate pre-clinical models for the evaluation of NMF in situations requiring periodontal or oral reconstruction. Here, we summarize commonly used pre-clinical models and provide examples of screening and functional studies of NMF that can be translated into clinical use.

Molded porous poly (L-lactide) membranes for guided bone regeneration with enhanced effects by controlled growth factor release

The aim of this study was to develop platelet-derived growth factor (PDGF-BB) loaded moldable porous poly (L-lactide) (PLLA)-tricalcium phosphate (TCP) membranes for guided bone regeneration (GBR) therapy. The membranes were designed to fit various types of bone defect sites. PDGF-BB-dissolved PLLA-TCP in methylene chloride-ethyl acetate solution was cast on a dome shaped metallic mold to fabricate a model membrane. The release rate of PDGF-BB, the osteoblast attachment test, and guided bone regeneration potential were evaluated with PDGF-BB-loaded PLLA-TCP membranes. Regular pores were generated throughout the membrane mainly due to phase inversion of PLLA-methylene chloride-ethyl acetate solution. A therapeutic amount of PDGF-BB was released from the membrane. The release rate could be controlled by varying the initial loading content of PDGF-BB. A significant amount of cells attached onto the PDGF-BB-loaded membrane rather than onto the unloaded membrane. Dome shaped bone formation was achieved in rabbit calvaria at 4 weeks. This indicated that restoration of bone defects to the bones original shape can be made possible by using molded membranes, which guide bone regeneration along with providing sufficient spaces. Bone forming efficiency was increased remarkably due to PDGF-BB release from PLLA-TCP membranes. These results suggested that the PDGF-BB releasing molded PLLA-TCP membrane may potentially improve GBR efficiency in various types of bone defects.

PDGF-B Gene Therapy Accelerates Bone Engineering and Oral Implant Osseointegration

Platelet-derived growth factor-BB (PDGF-BB) stimulates repair of healing-impaired chronic wounds such as diabetic ulcers and periodontal lesions. However, limitations in predictability of tissue regeneration occur due, in part, to transient growth factor bioavailability in vivo. Here, we report that gene delivery of PDGF-B stimulates repair of oral implant extraction socket defects. Alveolar ridge defects were created in rats and were treated at the time of titanium implant installation with a collagen matrix containing an adenoviral (Ad) vector encoding PDGF-B (5.5 × 108 or 5.5 × 109 pfu ml−1), Ad encoding luciferase (Ad-Luc; 5.5 × 109 pfu ml−1; control) or recombinant human PDGF-BB protein (rhPDGF-BB, 0.3 mg ml−1). Bone repair and osseointegration were measured through backscattered scanning electron microscopy, histomorphometry, micro-computed tomography and biomechanical assessments. Furthermore, a panel of local and systemic safety assessments was performed. Results indicated that bone repair was accelerated by Ad-PDGF-B and rhPDGF-BB delivery compared with Ad-Luc, with the high dose of Ad-PDGF-B more effective than the low dose. No significant dissemination of the vector construct or alteration of systemic parameters was noted. In summary, gene delivery of Ad-PDGF-B shows regenerative and safety capabilities for bone tissue engineering and osseointegration in alveolar bone defects comparable with rhPDGF-BB protein delivery in vivo.

Adenovirus Encoding Human Platelet-Derived Growth Factor-B Delivered to Alveolar Bone Defects Exhibits Safety and Biodistribution Profiles Favorable for Clinical Use

Platelet-derived growth factor (PDGF) gene therapy offers promise for tissue engineering of tooth-supporting alveolar bone defects. To date, limited information exists regarding the safety profile and systemic biodistribution of PDGF gene therapy vectors when delivered locally to periodontal osseous defects. The aim of this preclinical study was to determine the safety profile of adenovirus encoding the PDGF-B gene (AdPDGF-B) delivered in a collagen matrix to periodontal lesions. Standardized alveolar bone defects were created in rats, followed by delivery of matrix alone or containing AdPDGF-B at 5.5 x 10(8) or 5.5 x 10(9) plaque-forming units/ml. The regenerative response was confirmed histologically. Gross clinical observations, hematology, and blood chemistries were monitored to evaluate systemic involvement. Bioluminescence and quantitative polymerase chain reaction were used to assess vector biodistribution. No significant histopathological changes were noted during the investigation. Minor alterations in specific hematological and blood chemistries were seen; however, most parameters were within the normal range for all groups. Bioluminescence analysis revealed vector distribution at the axillary lymph nodes during the first 2 weeks with subsequent return to baseline levels. AdPDGF-B was well contained within the localized osseous defect area without viremia or distant organ involvement. These results indicate that AdPDGF-B delivered in a collagen matrix exhibits acceptable safety profiles for possible use in human clinical studies.

Novel three-dimensional scaffolds of poly(L-lactic acid) microfibers using electrospinning and mechanical expansion: Fabrication and bone regeneration

Poly(L-lactic acid) (PLLA) microfibrous scaffolds with three-dimensional (3D) structures were fabricated using an electrospinning technique with a subsequent mechanical expansion process. To achieve a 3D fibrous structure, the fusion at the contact points of the as-spun PLLA microfibers was avoided using an appropriate binary solvent system of methylene chloride and acetone. The solvent composition was optimized based on the solvent power, volatility, and viscosity (methylene chloride:acetone = 9:1 volume ratio). The final 3D structure of the electrospun scaffolds was obtained after mechanical expansion of the electrospun microfibrous mats. The pore sizes of the scaffolds were controlled by varying the degree of expansion of the nonbonded microfibrous mats, and they were in the range of several microns up to 400 μm. The 3D scaffolds were examined for their morphological properties and their potential use for the proliferation of osteoblasts. Generally recognized electrospun 2D nanofibrous membranes were also tested in order to compare the cell behaviors using different scaffold geometries. The 3D scaffolds demonstrated a high level of osteoblast proliferation (1.8-fold higher than nanofibrous membranes in a week). The osteoblasts actively penetrated the inside of the 3D scaffold and showed a spatial cell distribution, as confirmed by SEM and H&E staining, while a monolayer formed in the case of the 2D nanofibrous membranes with limited cell infiltration. In vivo results further showed that 3D electrospun microfibrous matrices were a favorable substrate for cell infiltration and bone formation after 2 and 4 weeks, using a rabbit calvarial defect model. In this study, the 3D microfibrous PLLA scaffolds fabricated using electrospinning techniques might be an innovative addition to tissue engineering applications.

Evaluations of osteogenic and osteoconductive properties of a non-woven silica gel fabric made by the electrospinning method

Evaluations of the osteoblast-like cell responses and osteoconductivity of a non-woven silica gel fabric were carried out to determine its potential for application as a scaffold material for use in bone tissue engineering. The silica gel solution was prepared by condensation following hydrolysis of tetraethyl orthosilicate under acidic conditions. The solution was spun under a 2kVcm(-1) electric field. The diameters of the as-spun silica gel fibers were in the range of approximately 0.7-6microm. The fabric was then heat-treated at 300 degrees C for 3h. The proliferation of pre-osteoblastic MC3T3-E1 cells evaluated by the MTS assay was lower than on the tissue culture plate (TCP) as many cells leaked through the large voids formed by the randomly placed long, narrow silica gel fibers, which further retarded cell growth. However, the expressions of extracellular signal-regulated kinase and transcriptional factor from the cells were higher when cultured on the non-woven silica gel fabrics than on TCP. The alkaline phosphatase (ALP) activity and differentiation marker expressions assessed by amplication via the reverse transcription-polymerase chain reaction, such as type I collagen, ALP and osteocalcin, were higher for cells cultured on non-woven silica gel fabrics than on TCP. The non-woven silica gel fabric showed good osteoconductivity in the calvarial defect New Zealand white rabbit model. To this end, the non-woven silica gel fabric has good potential as a scaffold material for bone tissue engineering due to its good biological properties.