Chih-Hao Chen

Incorporation of chitosan in biomimetic gelatin/chondroitin-6-sulfate/hyaluronan cryogel for cartilage tissue engineering

We prepare an elastic macroporous gelatin/chondoitin-6-sulfate/hyaluronan (GCH) cryogel scaffold mimic the composition of cartilage extracellular matrix for cartilage tissue engineering. By incorporating chitosan in the cryogel to replace 20% gelatin, a GCH-chitosan cryogel was also synthesized and compared with GCH cryogel for scaffold mechanical properties and chondrocytes response. The GCH-chitosan cryogel has larger pores, higher ultimate strain (stress) and elastic modulus, and lower stress relaxation percentage than the GCH cryogel. Both cryogels show a highly elastic property with a loss tangent around 0.1, but chitosan incorporation increases the storage modulus (elasticity). Chondrocytes proliferate and redifferentiate in cryogels; chitosan diminishes cell proliferation but up-regulates glycosaminoglycans (GAGs) and type II collagen (COL II) secretion. Implantation of a chondrocytes/GCH-chitosan cryogel construct in a full-thickness articular cartilage defect regenerates cartilage with positive stainings for GAGs and COL II and an elastic modulus similar to the native cartilage.

Surface modification of polycaprolactone scaffolds fabricated via selective laser sintering for cartilage tissue engineering

Surface modified porous polycaprolactone scaffolds fabricated via rapid prototyping techniques were evaluated for cartilage tissue engineering purposes. Polycaprolactone scaffolds manufactured by selective laser sintering (SLS) were surface modified through immersion coating with either gelatin or collagen. Three groups of scaffolds were created and compared for both mechanical and biological properties. Surface modification with collagen or gelatin improved the hydrophilicity, water uptake and mechanical strength of the pristine scaffold. From microscopic observations and biochemical analysis, collagen-modified scaffold was the best for cartilage tissue engineering in terms of cell proliferation and extracellular matrix production. Chondrocytes/collagen-modified scaffold constructs were implanted subdermally in the dorsal spaces of female nude mice. Histological and immunohistochemical staining of the retrieved implants after 8 weeks revealed enhanced cartilage tissue formation. We conclude that collagen surface modification through immersion coating on SLS-manufactured scaffolds is a feasible scaffold for cartilage tissue engineering in craniofacial reconstruction.

Combination of guided osteogenesis with autologous platelet-rich fibrin glue and mesenchymal stem cell for mandibular reconstruction.

BACKGROUNDnThis study examined whether a combination of autologous platelet-rich fibrin glue (PRFG) with mesenchymal stem cells (MSCs) and MEDPOR as guided tissue regeneration (GTR) could act as an osteogenic substitute and whether this treatment yields faster new bone formation than MEDPOR alone or PRFG plus MSC.nnnMATERIALnMSCs were harvested and isolated from the bone marrow of dog ilium. Full-thickness bony defects (1.5×1.5 cm) were created in the bilateral mandible angles of the dog. Treatments for bone defect in each group were as follows: group I (n=4), MEDPOR sheet as GTR and autologous PRFG/MSCs admixtures; group II (n=4), autologous PRFG/MSCs admixtures; group III (n=4), MEDPOR sheet as GTR; and group IV (n=4), control (empty defect). The percentage of new bone regeneration in computerized tomography at 2 months and 4 months was calculated by Analyze version 7.0 software. The mandibles were harvested from all specimens at 4 months, and the grafted sites were evaluated by gross, histologic, and X-ray examination.nnnRESULTSnBy radiographic analysis at 16 weeks posttransplantation, it was shown that an average of 72.8%±8.02% new bone formation in group I, 53.34%±6.87% in group II, 26.58%±6.41% in group III, and 15.14%±2.37% in group IV. Histologic examination revealed that the defect was repaired by typical bone tissue in groups I and II, whereas only minimal bone formation with fibrous connection was observed in the groups III and IV group. Besides, muscle incarceration was found in groups II and IV without MEDPOR as GTR.nnnCONCLUSIONnAutologous PRFG plus osteoinduced MSCs have good potential for bone regeneration. In combination with MEDPOR as GTR, bone regeneration is enhanced by preventing soft tissue ingrowth hindering bone regeneration.

Dual functional core–sheath electrospun hyaluronic acid/polycaprolactone nanofibrous membranes embedded with silver nanoparticles for prevention of peritendinous adhesion

Peritendinous adhesions, one of the common complications after tendon injury and subsequent surgery, could be minimized by directly placing a physical barrier between the injured site and the surrounding tissue. We used silver (Ag) nanoparticles embedded in electrospun hyaluronic acid (HA)/polycaprolactone (PCL) nanofibrous membranes (NFMs) (HA/PCL+Ag NFMs) to prevent peritendinous adhesions and bacterial infection after tendon surgery. HA was used for effective lubrication, and Ag provided antibacterial activity. A dual functional anti-adhesion barrier with core-sheath nanofibrous architecture was made from an HA core solution and a photo-reduced silver nitrate/PCL sheath solution. Polycaprolactone NFMs (PCL NFMs), hyaluronic acid/polycaprolactone core-sheath NFMs (HA/PCL NFMs) and HA/PCL+Ag NFMs with comparable fiber diameters and pore sizes were prepared and analyzed. The microporous structure of NFMs is expected to effectively block the penetration of adhesion-forming fibroblasts during tendon healing. The release of Ag from HA/PCL+Ag NFMs plateaued after 4 days, which confirmed the short-term anti-bacterial effect, and this result was verified with agar diffusion tests. In contrast, the release of HA was extended up to 21 days to simulate the lubrication effect offered by HA in the synovial fluid of the tendon sheath. In vitro cell culture experiments revealed that HA/PCL+Ag NFMs exhibited the highest inhibition of fibroblast attachment and proliferation without significant cytotoxicity due to the synergistic effect of Ag and HA. In vivo studies with a rabbit flexor tendon model further confirmed the efficacy of HA/PCL+Ag NFMs in reducing peritendinous adhesion as determined by gross observation, histology, joint range-of-motion, tendon gliding and biomechanical tests.

Cartilage Tissue Engineering with Silk Fibroin Scaffolds Fabricated by Indirect Additive Manufacturing Technology

Advanced tissue engineering (TE) technology based on additive manufacturing (AM) can fabricate scaffolds with a three-dimensional (3D) environment suitable for cartilage regeneration. Specifically, AM technology may allow the incorporation of complex architectural features. The present study involves the fabrication of 3D TE scaffolds by an indirect AM approach using silk fibroin (SF). From scanning electron microscopic observations, the presence of micro-pores and interconnected channels within the scaffold could be verified, resulting in a TE scaffold with both micro- and macro-structural features. The intrinsic properties, such as the chemical structure and thermal characteristics of SF, were preserved after the indirect AM manufacturing process. In vitro cell culture within the SF scaffold using porcine articular chondrocytes showed a steady increase in cell numbers up to Day 14. The specific production (per cell basis) of the cartilage-specific extracellular matrix component (collagen Type II) was enhanced with culture time up to 12 weeks, indicating the re-differentiation of chondrocytes within the scaffold. Subcutaneous implantation of the scaffold-chondrocyte constructs in nude mice also confirmed the formation of ectopic cartilage by histological examination and immunostaining.

Prevention of peritendinous adhesions with electrospun chitosan-grafted polycaprolactone nanofibrous membranes.

As one of the common complications after tendon injury and subsequent surgery, peritendinous adhesions could be minimized by directly placing a physical barrier between the injured site and the surrounding tissue. With the aim of solving the shortcomings of current biodegradable anti-adhesion barrier membranes, we propose the use of an electrospun chitosan-grafted polycaprolactone (PCL-g-CS) nanofibrous membrane (NFM) to prevent peritendinous adhesions. After introducing carboxyl groups on the surface by oxygen plasma treatment, the polycaprolactone (PCL) NFM was covalently grafted with chitosan (CS) molecules, with carbodiimide as the coupling agent. Compared with PCL NFM, PCL-g-CS NFM showed a similar fiber diameter, permeation coefficient for bovine serum albumin, ultimate tensile strain, reduced pore diameter, lower water contact angle, increased water sorption and tensile strength. With its submicrometer pore diameter (0.6-0.9μm), both NFMs could allow the diffusion of nutrients and waste while blocking fibroblast penetration to prevent adhesion formation after tendon surgery. Cell culture experiments verified that PCL-g-CS NFM can reduce fibroblast attachment while maintaining the biocompatibility of PCL NFM, implicating a synergistic anti-adhesion effect to raise the anti-adhesion efficacy. In vivo studies with a rabbit flexor digitorum profundus tendon surgery model confirmed that PCL-g-CS NFM effectively reduced peritendinous adhesion from gross observation, histology, joint flexion angle, gliding excursion and biomechanical evaluation. An injured tendon wrapped with PCL-g-CS NFM showed the same tensile strength as the naturally healed tendon, indicating that the anti-adhesion NFM will not compromise tendon healing.

Modulation of Bone-Specific Tissue Regeneration by Incorporating Bone Morphogenetic Protein and Controlling the Shell Thickness of Silk Fibroin/Chitosan/Nanohydroxyapatite Core-Shell Nanofibrous Membranes.

The presence of both osteoconductive and osteoinductive factors is important in promoting stem cell differentiation toward the osteogenic lineage. In this study, we prepared silk fibroin/chitosan/nanohydroxyapatite/bone morphogenetic protein-2 (SF/CS/nHAP/BMP-2, SCHB2) nanofibrous membranes (NFMs) by incorporating BMP-2 in the core and SF/CS/nHAP as the shell layer of a nanofiber with two different shell thicknesses (SCHB2-thick and SCHB-thin). The physicochemical properties of SCHB2 membranes were characterized and compared with those of SF/CS and SF/CS/nHAP NFMs. When tested in release studies, the release rate of BMP-2 and the concentration of BMP-2 in the release medium were higher for SCHB2-thin NFMs because of reduced shell thickness. The BMP-2 released from the nanofiber retained its osteoinductive activity toward human-bone-marrow-derived mesenchymal stem cells (hMSCs). Compared with SF/CS and SF/CS/nHAP NFMs, the incorporation of BMP-2-promoted osteogenic differentiation of hMSCs and the SCHB-thin NFM is the best scaffold during in vitro cell culture. Gene expression analysis by real-time quantitative polymerase chain reaction detected the evolution of both early and late marker genes of bone formation. The relative mRNA expression is in accordance with the effect of BMP-2 incorporation and shell thickness, while the same was reconfirmed through the quantification of bone marker protein osteocalcin. In vivo experiments were carried out by subcutaneously implanting hMSC-seeded SCHB2-thin NFMs and acellular controls on the back sides of nude mice. Immunohistochemical and histological staining confirmed ectopic bone formation and osteogenesis of hMSCs in SCHB2-thin NFMs. In conclusion, the SCHB2-thin NFM could be suggested as a promising scaffold for bone tissue engineering.

Solvent-free fabrication of three dimensionally aligned polycaprolactone microfibers for engineering of anisotropic tissues

Fabrication of aligned microfiber scaffolds is critical in successful engineering of anisotropic tissues such as tendon, ligaments and nerves. Conventionally, aligned microfiber scaffolds are two dimensional and predominantly fabricated by electrospinning which is solvent dependent. In this paper, we report a novel technique, named microfiber melt drawing, to fabricate a bundle of three dimensionally aligned polycaprolactone microfibers without using any organic solvent. This technique is simple yet effective. It has been demonstrated that polycaprolactone microfibers of 10xa0μm fiber diameter can be directly drawn from a 2xa0mm orifice. Orifice diameter, temperature and take-up speed significantly influence the final linear density and fiber diameter of the microfibers. Mechanical test suggests that mechanical properties such as stiffness and breaking force of microfiber bundles can be easily adjusted by the number of fibers. In vitro study shows that these microfibers are able to support the proliferation of human dermal fibroblasts over 7xa0days. In vivo result of Achilles tendon repair in a rabbit model shows that the microfibers were highly infiltrated by tendon tissue as early as in 1xa0month, besides, the repaired tendon have a well-aligned tissue structure under the guidance of aligned microfibers. However whether these three dimensionally aligned microfibers can induce three dimensionally aligned cells remains inconclusive.

Selective laser sintered poly-ε-caprolactone scaffold hybridized with collagen hydrogel for cartilage tissue engineering.

Selective laser sintering (SLS), an additive manufacturing (AM) technology, can be used to produce tissue engineering scaffolds with pre-designed macro and micro features based on computer-aided design models. An in-house SLS machine was built and 3D poly-ε-caprolactone (PCL) scaffolds were manufactured using a layer-by-layer design of scaffold struts with varying orientations (0°/45°/0°/45°, 0°/90°/0°/90°, 0°/45°/90°/135°), producing scaffolds with pores of different shapes and distribution. To better enhance the scaffold properties, chondrocytes were seeded in collagen gel and loaded in scaffolds for cartilage tissue engineering. Gel uptake and dynamic mechanical analysis demonstrated the better suitability of the 0°/90°/0°/90° scaffolds for reconstructive cartilage tissue engineering purposes. Chondrocytes were then seeded onto the 0°/90°/0°/90° scaffolds in collagen I hydrogel (PCL/COL1) and compared to medium-suspended cells in terms of their cartilage-like tissue engineering parameters. PCL/COL1 allowed better cell proliferation when compared to PCL or two-dimensional tissue culture polystyrene. Scanning electron microscopy and confocal microscopy observations demonstrated a similar trend for extracellular matrix production and cell survival. Glycosaminoglycan and collagen II quantification also demonstrated the superior matrix secretion properties of PCL/COL1 hybrid scaffolds. Collagen-gel-suspended chondrocytes loaded in SLS-manufactured PCL scaffolds may provide a means of producing tissue-engineered cartilage with customized shapes and designs via AM technology.

Preparation and characterization of antiadhesion barrier film from hyaluronic acid-grafted electrospun poly(caprolactone) nanofibrous membranes for prevention of flexor tendon postoperative peritendinous adhesion.

Peritendinous adhesion is one of the common complications encountered after tendon injury and subsequent surgery, and it can be minimized by introducing a physical barrier between the injured site and the surrounding tissue. An electrospun hyaluronic acid-grafted poly(caprolactone) (PCL-g-HA) nanofibrous membrane (NFM) is proposed as an alternative to current antiadhesion barrier films. HA is covalently grafted to surface-aminolyzed PCL nanofibers, using carbodiimide as the coupling agent. Pristine PCL and PCL-g-HA NFMs were characterized by scanning electron microscopy, thermogravimetric analysis, X-ray photoelectron spectroscopy, Fourier-transform infrared spectroscopy, and mechanical testing. In vitro cell culture with fibroblasts showed that PCL-g-HA NFMs reduced cellular adhesion on the membrane surface while maintaining cell proliferation. Animal experiments using a rabbit flexor digitorum profundus tendon model confirmed the efficacy of PCL-g-HA in reducing peritendinous adhesion, based on gross observation, histology, joint flexion-angle measurements, gliding tests, and biomechanical evaluation.