Incorporating the Antioxidant Fullerenol into Calcium Phosphate Bone Cements Increases Cellular Osteogenesis without Compromising Physical Cement Characteristics.

Herein, fullerenol (Ful), a highly water-soluble derivative of C60 fullerene with demonstrated antioxidant activity, is incorporated into calcium phosphate cements (CPCs) to enhance their osteogenic ability. CPCs with added carboxymethyl cellulose/gelatin (CMC/Gel) are doped with biocompatible Ful particles at concentrations of 0.02, 0.04, and 0.1 wt v%-1 and evaluated for Ful-mediated mechanical performance, antioxidant activity, and in vitro cellular osteogenesis. CMC/gel cements with the highest Ful concentration decrease setting times due to increased hydrogen bonding from Ful's hydroxyl groups. In vitro studies of reactive oxygen species (ROS) scavenging with CMC/gel cements demonstrate potent antioxidant activity with Ful incorporation and cement scavenging capacity is highest for 0.02 and 0.04 wt v%-1 Ful. In vitro cytotoxicity studies reveal that 0.02 and 0.04 wt v%-1 Ful cements also protect cellular viability. Finally, increase of alkaline phosphatase (ALP) activity and expression of runt-related transcription factor 2 (Runx2) in MC3T3-E1 pre-osteoblast cells treated with low-dose Ful cements demonstrate Ful-mediated osteogenic differentiation. These results strongly indicate that the osteogenic abilities of Ful-loaded cements are correlated with their antioxidant activity levels. Overall, this study demonstrates exciting potential of Fullerenol as an antioxidant and proosteogenic additive for improving the performance of calcium phosphate cements in bone reconstruction procedures.

Surface analysis of (Ti,Mg)N coated bone fixation devices following the rabbit femur surgery.

BACKGROUND: Magnesium (Mg) enhances the bone regeneration, mineralization and attachment at the tissue/biomaterial interface. OBJECTIVE: In this study, the effect of Mg on mineralization/osseointegration was determined using (Ti,Mg)N thin film coated Ti6Al4V based plates and screws in vivo. METHODS: TiN and (Ti,Mg)N coated Ti6Al4V plates and screws were prepared using arc-PVD technique and used to fix rabbit femur fractures for 6 weeks. Then, mineralization/osseointegration was assessed by surface analysis including cell attachment, mineralization, and hydroxyapatite deposition on concave and convex sides of the plates along with the attachment between the screw and the bone. RESULTS: According to Scanning Electron Microscopy (SEM) and Energy Dispersive Spectroscopy (EDS) analyses; cell attachment and mineralization were higher on the concave sides of the plates from both groups in comparison to the convex sides. However, mineralization was significantly higher on Mg-containing ones. The mean gray value indicating mineralized area after von Kossa staining was found as 0.48 ± 0.01 and 0.41 ± 0.04 on Mg containing and free ones respectively. Similarly, Fourier Transform Infrared Spectroscopy (FTIR) and X-ray diffraction (XRD) analyses showed that hydroxyapatite growth was abundant on the Mg-containing and concave sides of the plates. Enhanced mineralization and strong attachment to bone were also detected in EDS and SEM analyses of Mg-containing screws. CONCLUSION: These findings indicated that (Ti,Mg)N coatings can be used to increase attachment at the implant tissue interface due to accelerated mineralization, cell attachment, and hydroxyapatite growth.

Chondro-inductive hyaluronic acid/chitosan coacervate-based scaffolds for cartilage tissue engineering.

Injuries related to articular cartilage are among the most challenging musculoskeletal problems because of poor repair capacity of this tissue. The lack of efficient treatments for chondral defects has stimulated research on cartilage tissue engineering applications combining porous biocompatible scaffolds with stem cells in the presence of external stimuli. This work presents the role of rat bone marrow mesenchymal stem cell (BMSC) encapsulated-novel three-dimensional (3D) coacervate scaffolds prepared through complex coacervation between different chitosan salts (CHI) and sodium hyaluronate (HA). The 3D architecture of BMSC encapsulated scaffolds (HA/CHI) was shown by scanning electron microscopy (SEM) to have an interconnected structure to allow cell-cell and cell-matrix interactions. Chondrogenic induction of encapsulated BMSCs within HA/CHI coacervates demonstrated remarkable cellular viability in addition to the elevated expression levels of chondrogenic markers such as sex determining region Y-box 9 protein (SOX9), aggrecan (ACAN), cartilage oligomeric matrix protein (COMP) and collagen type II (COL2A1) by immunofluorescence staining, qPCR and ELISA test. Collectively, HA/CHI coacervates are promising candidates for future use of these scaffolds in cartilage tissue engineering applications.

Assessment of bone healing using (Ti,Mg)N thin film coated plates and screws: Rabbit femur model.

Magnesium (Mg) based implants such as plates and screws are often preferred to treat bone defects because of the positive effects of magnesium in bone growth and healing. Their low corrosion resistance, however, leads to fast degradation and consequently failure before healing was completed. Previously, we developed Mg doped titanium nitrate (TiN) thin film coatings to address these limitations and demonstrated that <10 at% Mg doping led to enhanced mineralization in vitro. In the present study, in vivo performance of (Ti,Mg)N coated Ti6Al4V based plates and screws were studied in the rabbit model. Bone fractures were formed on femurs of 16 rabbits and then fixed with either (Ti,Mg)N coated (n = 8) or standard TiN coated (n = 8) plates and screws. X-ray imaging and µCT analyses showed enhanced bone regeneration on fracture sites fixed with (Ti,Mg)N coated plates in comparison with the Mg free ones. Bone mineral density, bone volume, and callus volume were also found to be 11.4, 23.4, and 42.8% higher, respectively, in accordance with µCT results. Furthermore, while TiN coatings promoted only primary bone regeneration, (Ti,Mg)N led to secondary bone regeneration in 6 weeks. These results indicated that Mg presence in the coatings accelerated bone regeneration in the fracture site. (Ti,Mg)N coating can be used as a practical method to increase the efficiency of existing bone fixation devices of varying geometry.

Microfibrous scaffolds from poly(l-lactide-co-ε-caprolactone) blended with xeno-free collagen/hyaluronic acid for improvement of vascularization in tissue engineering applications.

Success of 3D tissue substitutes in clinical applications depends on the presence of vascular networks in their structure. Accordingly, research in tissue engineering is focused on the stimulation of angiogenesis or generation of a vascular network in the scaffolds prior to implantation. A novel, xeno-free, collagen/hyaluronic acid-based poly(l-lactide-co-ε-caprolactone) (PLC/COL/HA) (20/9.5/0.5 w/w/w) microfibrous scaffold was produced by electrospinning. Collagen types I and III, and hyaluronic acid were isolated from human umbilical cords and blended with the GMP grade PLC. When compared with PLC scaffolds the PLC/COL/HA had higher water uptake capacity (103% vs 66%) which may have contributed to the decrease in its Young's Modulus (from 1.31 to 0.89 MPa). The PLC/COL/HA better supported adipose tissue-derived mesenchymal stem cell (AT MSC) adhesion; within 24 h the cell number on the PLC/COL/HA scaffolds was 3 fold higher. Co-culture of human umbilical vein endothelial cells and AT MSCs induced capillary formation on both scaffold types, but the PLC/COL/HA led to formation of interconnected vessels whose total length was 1.6 fold of the total vessel length on PLC. Clinical use of this scaffold would eliminate the immune response triggered by xenogeneic collagen and transmission of animal-borne diseases while promoting a better vascular network formation.

Gene Therapy Strategies in Bone Tissue Engineering and Current Clinical Applications.

Gene therapy provides a promising approach for regeneration and repair of injured bone. Application of gene therapy has displayed increased efficiency in various animal models and preclinical trials in comparison with traditional bone grafting methods. The objective of this review is to highlight fundamental principles of gene therapy strategies in bone tissue engineering and solutions of their current limitations for the healing of bone injury. Vector types are debated for the repair of defected site due to demonstration of constraints and applications of the protocols. In recent years, the combination of gene therapy strategies and bone tissue engineering has highly gained attention. We discussed viral and non-viral mediated delivery of therapeutic protein by using scaffolds for bone tissue engineering. Although pre-clinical studies have showed that gene therapy has very promising results to heal injured bone, there are several limitations regarding with the usage of gene delivery methods into clinical applications. Choice of suitable vector, selection of transgene and gene delivery protocols are the most outstanding questions. This article also addresses current state of gene delivery strategies in bone tissue engineering for their potential applications in clinical considerations.

Dental Stem Cells in Bone Tissue Engineering: Current Overview and Challenges.

The treatment of bone that is impaired due to disease, trauma or tumor resection creates a challenge for both clinicians and researchers. Critical size bone defects are conventionally treated with autografts which are associated with risks such as donor site morbidity and limitations like donor shortage. Bone tissue engineering has become a promising area for the management of critical size bone defects by the employment of biocompatible materials and the discovery of novel stem cell sources. Mesenchymal stem cells (MSCs) can be isolated with ease from various dental tissues including dental pulp stem cells, stem cells from apical papilla, dental follicle stem cells, stem cells from human exfoliated deciduous teeth, periodontal ligament stem cells, gingival stem cells and tooth germ derived stem cells. Outcomes of dental MSC mediated bone tissue engineering is explored in various in vivo and in vitro preclinical studies. However, there are still obscurities regarding the mechanisms underlying in MSC mediated bone regeneration and challenges in applications of dental stem cells. In this review, we summarized dental stem cell sources and their characterizations, along with currently used biomaterials for cell delivery and future perspectives for dental MSCs in the field of bone tissue engineering. Further efforts are necessary before moving to clinical trials for future applications.

Bone Formation from Porcine Dental Germ Stem Cells on Surface Modified Polybutylene Succinate Scaffolds.

Designing and providing a scaffold are very important for the cells in tissue engineering. Polybutylene succinate (PBS) has high potential as a scaffold for bone regeneration due to its capacity in cell proliferation and differentiation. Also, stem cells from 3rd molar tooth germs were favoured in this study due to their developmentally and replicatively immature nature. In this study, porcine dental germ stem cells (pDGSCs) seeded PBS scaffolds were used to investigate the effects of surface modification with fibronectin or laminin on these scaffolds to improve cell attachment, proliferation, and osteogenic differentiation for tissue engineering applications. The osteogenic potentials of pDGSCs on these modified and unmodified foams were examined to heal bone defects and the effects of fibronectin or laminin modified PBS scaffolds on pDGSC differentiation into bone were compared for the first time. For this study, MTS assay was used to assess the cytotoxic effects of modified and unmodified surfaces. For the characterization of pDGSCs, flow cytometry analysis was carried out. Besides, alkaline phosphatase (ALP) assay, von Kossa staining, real-time PCR, CM-Dil, and immunostaining were applied to analyze osteogenic potentials of pDGSCs. The results of these studies demonstrated that pDGSCs were differentiated into osteogenic cells on fibronectin modified PBS foams better than those on unmodified and laminin modified PBS foams.

Targeted mesenchymal stem cell and vascular endothelial growth factor strategies for repair of nerve defects with nerve tissue implanted autogenous vein graft conduits.

Peripheral nerve gaps exceeding 1 cm require a bridging repair strategy. Clinical feasibility of autogenous nerve grafting is limited by donor site comorbidity. In this study we investigated neuroregenerative efficacy of autogenous vein grafts implanted with tissue fragments from distal nerve in combination with vascular endothelial growth factor (VEGF) or mesenchymal stem cells (MSCs) in repair of rat peripheral nerve defects. Six-groups of Sprague-Dawley rats (n = 8 each) were evaluated in the autogenous setting using a 1.6 cm long peroneal nerve defect: Empty vein graft (group 1), Nerve graft (group 2), Vein graft and nerve fragments (group 3), Vein graft and nerve fragments and blank microspheres (group 4), Vein graft and nerve fragments and VEGF microspheres (group 5), Vein graft and nerve fragments and MSCs (group 6). Nerve fragments were derived from distal segment. Walking track analysis, electrophysiology and nerve histomorphometry were performed for assessment. Peroneal function indices (PFI), electrophysiology (amplitude) and axon count results for group 2 were -9.12 ± 3.07, 12.81 ± 2.46 mV, and 1697.88 ± 166.18, whereas the results for group 5 were -9.35 ± 2.55, 12.68 ± 1.78, and 1566 ± 131.44, respectively. The assessment results did not reveal statistical difference between groups 2 and 5 (P > 0.05). The best outcomes were seen in group 2 and 5 followed by group 6. Compared to other groups, poorest outcomes were seen in group 1 (P ≤ 0.05). PFI, electrophysiology (amplitude) and axon count results for group 1 were -208.82 ± 110.69, 0.86 ± 0.52, and 444.50 ± 274.03, respectively. Vein conduits implanted with distal nerve-derived nerve fragments improved axonal regeneration. VEGF was superior to MSCs in facilitating nerve regeneration. © 2015 Wiley Periodicals, Inc. Microsurgery 36:578-585, 2016.

Response of CD44+/CD24-/low breast cancer stem/progenitor cells to tamoxifen­ and doxorubicin­induced autophagy.

The cancer stem cell hypothesis emphasizes that cancers are driven by cells having stem cell properties, and it is believed that cancer stem cells (CSCs) may be responsible for resistance against therapeutic approaches and for recurrent tumors. Since the biology of the normal breast requires large numbers of stem cells, it has been thought that breast stem cells play an important role in initiating breast cancer. A better characterization of breast CSCs appears to be an essential step to improve the understanding of the biology of breast cancer and its management. The scope of this study was to isolate breast CSCs from a breast cancer cell line (MCF-7) using cell surface markers, and to test whether these cells have any resistance to autophagic cell death mechanisms mediated by commonly used chemotherapies and hormonal therapies such as doxorubicin (adriamycin) and tamoxifen (anti-estrogen), respectively. For this purpose, the CD44+/CD24-/low MCF-7 breast cancer stem/progenitor cell population was isolated and treated with doxorubicin or tamoxifen and evaluated for their response to growth, autophagy and apoptosis. Our findings suggest that CD44+/CD24-/low cells were less sensitive to doxorubicin, but did not demonstrate a significant difference towards tamoxifen in regards to the induction of autophagy.

The effect of subcutaneous mesenchymal stem cell injection on statis zone and apoptosis in an experimental burn model.

BACKGROUND: In an acute burn injury the zone of stasis is initially vital but may progress to coagulation necrosis with time. In this study, salvage of the zone of stasis was aimed at by subcutaneous mesenchymal stem cell injection. METHODS: Mesenchymal stem cells were obtained from the bone marrow of Sprague-Dawley rats (n = 10). Twenty Sprague-Dawley rats received thermal injury on the back according to the previously described "comb burn" model. Thirty minutes after the burn injury, mesenchymal stem cells were injected subcutaneously to the stasis zone of the experimental group (n = 10). Animals in the control group (n = 10) were given the same amount of saline without mesenchymal stem cells. Animals in the sham group (n = 6) did not receive any thermal trauma. Seventy-two hours after the burn injury, scintigraphic examination was applied to determine average vital tissue at the stasis zone. Thereafter, skin samples were assessed by immunohistochemistry assay for apoptosis count. The blood samples drawn before and 72 hours after the burn injury were analyzed to determine systemic cytokine levels. RESULTS: The apoptosis count of the control group was found to be significantly higher than that of the experimental group. Vital tissue percentage of the stasis zone was significantly higher for the experimental group than for the control group. The cytokine levels did not reveal any statistically significant difference between the groups. CONCLUSION: Apoptosis count and scintigraphic results of this study confirm that mesenchymal stem cell treatment has a statistically significant benefit for the survival of the stasis zone in acute burn.

Bone response to biomimetic implants delivering BMP-2 and VEGF: an immunohistochemical study.

This animal study evaluated bone healing around titanium implant surfaces biomimetically coated with bone morphogenic protein-2 (BMP-2) and/or vascular endothelial growth factor (VEGF) by examining bone matrix proteins and mineralisation. Five different implant surfaces were established: acid-etched surface (AE), biomimetic calcium phosphate surface (CAP), BMP-2 loaded CAP surface, VEGF loaded CAP surface and dual BMP-2 + VEGF loaded CAP surface. The implants were inserted into calvariae of adult domestic pigs. For the comparison of osteoconductive capacity of each surface, bone mineral density and expression of bone matrix proteins (collagen I, BMP-2/4, osteocalcin and osteopontin) inside defined chambers around the implant were assessed using light microscopy and microradiography and immunohistochemical analysis at 1, 2 and 4 weeks. In the both groups delivering BMP-2, the bone mineral density was significantly enhanced after 2 weeks and the highest value was measured for the group BMP + VEGF. In the group VEGF, collagen I and BMP-2/4 expressions were significantly up-regulated at the first and second weeks. The percentage of BMP-2/4 positive cells in the group BMP + VEGF was significantly enhanced compared with the groups AE and CAP at the second week. Although the highest osteocalcin and osteopontin expression values were observed for the group BMP + VEGF after 2 weeks, no statistically significant difference in osteocalcin and osteopontin expressions was found between all groups at any time. It was concluded that combined delivery of BMP-2 and VEGF favoured bone mineralisation and expression of important bone matrix proteins that might explain synergistic interaction between both growth factors.

Effect of double growth factor release on cartilage tissue engineering.

The effects of double release of insulin-like growth factor I (IGF-I) and growth factor ß1 (TGF-ß1) from nanoparticles on the growth of bone marrow mesenchymal stem cells and their differentiation into cartilage cells were studied on PLGA scaffolds. The release was achieved by using nanoparticles of poly(lactic acid-co-glycolic acid) (PLGA) and poly(N-isopropylacrylamide) (PNIPAM) carrying IGF-I and TGF-ß1, respectively. On tissue culture polystyrene (TCPS), TGF-ß1 released from PNIPAM nanoparticles was found to have a significant effect on proliferation, while IGF-I encouraged differentiation, as shown by collagen type II deposition. The study was then conducted on macroporous (pore size 200-400 µm) PLGA scaffolds. It was observed that the combination of IGF-I and TGF-ß1 yielded better results in terms of collagen type II and aggrecan expression than GF-free and single GF-containing applications. It thus appears that gradual release of a combination of growth factors from nanoparticles could make a significant contribution to the quality of the engineered cartilage tissue.

Characterization of cancer stem-like cells in chordoma.

OBJECT: Chordomas are locally aggressive bone tumors known to arise from the remnants of the notochord. Because chordomas are rare, molecular studies aimed at developing new therapies are scarce and new approaches are needed. Chordoma cells and cancer stem-like cells share similar characteristics, including self-renewal, differentiation, and resistance to chemotherapy. Therefore, it seems possible that chordomas might contain a subpopulation of cancer stem-like cells. The aim of this study is to determine whether cancer stem-like cells might be present in chordomas. METHODS: In this study, the authors used gene expression analysis for common cancer stem-like cellmarkers, including c-myc, SSEA-1, oct4, klf4, sox2, nanog, and brachyury, and compared chordoma cells and tissues with nucleus pulposus tissues (disc degenerated nontumorigenic tissues). Differentiation through agents such as all-trans retinoic acid and osteogenic differentiation medium was induced to the chordoma cells. Additionally, U-CH1 cells were sorted via magnetic cell sorting for stem cell markers CD133 and CD15. After separation, positive and negative cells for these markers were grown in a nonadherent environment, soft agar, to determine whether the presence of these cancer stem-like cells might be responsible for initiating chordoma. The results were compared with those of untreated cells in terms of migration, proliferation, and gene expression by using reverse transcriptase polymerase chain reaction. RESULTS: The results indicate that chordoma cells might be differentiating and committing into an osteogenic lineage when induced with the osteogenic differentiation agent. Chordoma cells that are induced with retinoic acid showed slower migration and proliferation rates when compared with the untreated cells. Chordoma cells that were found to be enriched by cancer stem-like cell markers, namely CD133 and CD15, were able to live in a nonadherent soft agar medium, demonstrating a self-renewal capability. To the authors' knowledge, this is the first time that cancer stem-like cell markers were also found to be expressed in chordoma cells and tissues. CONCLUSIONS: Cancer stem-like cell detection might be an important step in determining the recurrent and metastatic characteristics of chordoma. This finding may lead to the development of new approaches toward treatments of chordomas.

Prefabrication of vascularized bone graft using an interconnected porous calcium hydroxyapatite ceramic in presence of vascular endothelial growth factor and bone marrow mesenchymal stem cells: Experimental study in rats.

OBJECTIVES: The purpose of this experimental pilot study was to create a prefabricated vascularized bone graft using interconnected porous calcium hydroxyapatite ceramic (PCHC) block by combining vascular bundle implantation, rat bone marrow mesenchymal stem cells and administration of vascular endothelial growth factor (VEGF) in a rat model. MATERIALS AND METHODS: Sixty male Sprague-Dawley rats were used. Experimental animals were divided into six groups, each of which comprised 10 rats. The PCHC blocks were implanted in the medial thigh region in groups I, III, and V without vascular bundle implantation. The PCHC blocks were vascularized by the superficial inferior epigastric artery and vein in groups II, IV and VI. These vessels were passed through the hole of the PCHC blocks. Mesenchymal stem cells were administered into the PCHC in groups III, IV, V and VI. In addition, both mesenchymal stem cells and VEGF were administered in group V and VI. The presence and density of any new bone formation and neovascularization from the vascular bundle was evaluated by X-ray, microangiography, scintigraphy, biochemical analysis and histomorphometry. RESULTS: The newly formed vessels and bone formations were significantly greater in group VI, in which both mesenchymal stem cells and VEGF were applied. CONCLUSION: THIS PRELIMINARY STUDY SUGGESTS THAT: Both mesenchymal stem cells and VEGF provide vascularized bone prefabrication by enhancing neovascularization and osteogenesis in a shorter time compared to only VEGF application.

Tissue engineered, guided nerve tube consisting of aligned neural stem cells and astrocytes.

Injury of the nervous system, particularly in the spinal cord, impairs the quality of life of the patient by resulting in permanent loss of neurologic function. The main limitation in spinal cord regeneration is the lack of extracellular matrix to guide nerves for functional recovery of the transected nerve tissue. In the present study, a tissue engineered nerve tube was prepared by wrapping neural stem cells (NSCs) on aligned fibers using a micropatterned film with astrocytes aligned along the microgrooves to support the NSCs. Initially the cell behavior on micropatterns and parallel fibers was investigated with cytoskeletal and nuclear staining, immunocytochemistry, and proliferation assay using the fiber and the film system separately. The results showed that both cells, NSCs in undifferentiated and astrocytes in differentiated form, were oriented in the direction of the guiding and support elements, the microgrooves, and the microfibers. They were able to grow and increase in number on these cell carriers. This trend was also maintained after the components were brought together in a nerve tube form and testing in coculture. The cells were able to survive and maintained their orientation in the 3D tissue engineered construct. The guided nerve tissue engineering approach tested in the present study with parallel NSCs and support cells in the tubular construct is expected to provide an appropriate environment for nerve regeneration in vivo.

Polyester based nerve guidance conduit design.

Nerve conduits containing highly aligned architecture that mimics native tissues are essential for efficient regeneration of nerve injuries. In this study, a biodegradable nerve conduit was constructed by converting a porous micropatterned film (PHBV-P(L-D,L)LA-PLGA) into a tube wrapping aligned electrospun fibers (PHBV-PLGA). The polymers were chosen so that the protective tube would erode slower than the fibrous core to achieve complete healing before the tube eroded. The pattern dimensions and the porosity (58.95 (%) with a maximum pore size of 4-5 microm) demonstrated that the micropatterned film would enable the migration, alignment and survival of native cells for proper regeneration. This film had sufficiently high mechanical properties (ultimate tensile strength: 3.13 MPa, Young's Modulus: 0.08 MPa) to serve as a nerve guide. Electrospun fibers, the inner part of the tubular construct, were well aligned with a fiber diameter of ca. 1.5 microm. Fiber properties were especially influenced by polymer concentration. SEM showed that the fibers were aligned parallel to the groove axis of the micropatterned film within the tube as planned considering the nerve tissue architecture. This two component nerve conduit appears to have the right organization for testing in vitro and in vivo nerve tissue engineering studies.

Tissue engineering of bone on micropatterned biodegradable polyester films.

In this study, the effect of cell alignment on proliferation and phenotype expression of rat bone marrow derived osteoblasts on micropatterned (MP) PHBV and P(L/D,L)LA films with 27 microm wide parallel microgrooves was investigated. Immobilization of fibrinogen (Fb) on film surface by adsorption increased hydrophilicity, while covalent immobilization decreased it. Amount of Fb immobilized was significantly higher upon covalent bonding (153.1+/-42.4 microg Fb/cm2) than when adsorbed (10.0+/-3.3 microg Fb/cm2). It was observed that the presence of MP did not influence cell proliferation in the long run. Osteoblasts on MP films with adsorbed (MP Fb(a)) and covalently immobilized Fb (MP Fb(i)) aligned parallel to the groove axis with mean deviation angles of 10.59+/-23.47 and 29.02+/-33.03 degrees, respectively, while on tissue culture polystyrene (TCP), on unpatterned films (UNP) and on UNP with adsorbed Fb (UNP Fb(a)) alignment with an arbitrary axis was much higher: 46.66+/-24.98, 48.72+/-31.19, 47.74+/-27.29 degrees, respectively. Fb-free MP films were not effective in cell alignment, and clumps were formed. Cell alignment achieved on MP Fb(a) films did not influence cell proliferation, but increased differentiation, as shown by ALP activity per cell and the evenness and the amount of calcium phosphate deposition. It was concluded that orientation of cells was influential on their differentiation and also, MP cell carriers with chemical cues on their surfaces are important in improving tissue repair.

Tissue engineered cartilage on collagen and PHBV matrices.

Cartilage engineering is a very novel approach to tissue repair through use of implants. Matrices of collagen containing calcium phosphate (CaP-Gelfix), and matrices of poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV) were produced to create a cartilage via tissue engineering. The matrices were characterized by scanning electron microscopy (SEM) and electron diffraction spectroscopy (EDS). Porosity and void volume analysis were carried out to characterize the matrices. Chondrocytes were isolated from the proximal humerus of 22 week-old male, adult, local albino rabbits. For cell type characterization, Type II collagen was measured by Western Blot analysis. The foams were seeded with 1x10(6) chondrocytes and histological examinations were carried out to assess cell-matrix interaction. Macroscopic examination showed that PHBV (with or without chondrocytes) maintained its integrity for 21 days, while CaP-Gelfix was deformed and degraded within 15 days. Cell-containing and cell-free matrices were implanted into full thickness cartilage defects (4.5 mm in diameter and 4 mm in depth) at the patellar groove on the right and left knees of eight rabbits, respectively. In vivo results at 8 and 20 weeks with chondrocyte seeded PHBV matrices presented early cartilage formation resembling normal articular cartilage and revealed minimal foreign body reaction. In CaP-Gelfix matrices, fibrocartilage formation and bone invasion was noted in 20 weeks. Cells maintained their phenotype in both matrices. PHBV had better healing response than CaP-Gelfix. Both matrices were effective in cartilage regeneration. These matrices have great potential for use in the repair of joint cartilage defects.

In vivo tissue engineering of bone using poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) and collagen scaffolds.

Porous poly(3-hydroxybutyric acid-co-3-hydroxyvaleric acid) (PHBV) and calcium phosphate-loaded collagen (CaP-Gelfix) foams were seeded with rat bone marrow stromal cells and implanted into defects created in rat femurs to study in vivo bone formation and to test their suitability for use in bone tissue engineering. At 3 and 6 weeks, new bone formation was evaluated by macroscopy, radiography, dual-energy X-ray absorptiometry (DEXA), and quantitative computerized tomography (QCT). Atomic contents of the implants were further assessed by QCT. Some initial inflammation that significantly decreased with time was observed in the CaP-Gelfix group. PHBV inflammation was minimal at all stages. Fibrous tissue formation in the CaP-Gelfix group was more than in the PHBV group. Both cell-loaded and cell-free PHBV matrices elicited minimal fibrous tissue formation during the 6-week implantation duration. Macroscopic and radiological studies demonstrated better healing with PHBV matrices than with CaP-Gelfix in 3 weeks. Histologically, fibrous connective tissue establishment and inflammation scores were significantly higher in the CaP-Gelfix group when compared with the PHBV group at both time intervals. At 6 weeks, however, the extent of healing was almost the same with both implants. DEXA and QCT results indicated that there was an increase in bone mineral density in both PHBV and CaP-Gelfix implants at the end of 6 weeks. This study suggests that even though PHBV and CaP-Gelfix have different bulk and surface chemistries they both are promising cell carriers that may be suitable for use in bone tissue engineering.