Showing papers in "Journal of Tissue Engineering and Regenerative Medicine in 2014"

Growth factors engineered for super-affinity to extracellular matrix enhance tissue healing

Abstract: Reference EPFL-CONF-201372View record in Web of Science Record created on 2014-08-29, modified on 2017-12-18

In vivo cartilage repair using adipose‐derived stem cell‐loaded decellularized cartilage ECM scaffolds

Abstract: We have previously reported a natural, human cartilage ECM (extracellular matrix)-derived three-dimensional (3D) porous acellular scaffold for in vivo cartilage tissue engineering in nude mice. However, the in vivo repair effects of this scaffold are still unknown. The aim of this study was to further explore the feasibility of application of cell-loaded scaffolds, using autologous adipose-derived stem cells (ADSCs), for cartilage defect repair in rabbits. A defect 4 mm in diameter was created on the patellar groove of the femur in both knees, and was repaired with the chondrogenically induced ADSC-scaffold constructs (group A) or the scaffold alone (group B); defects without treatment were used as controls (group C). The results showed that in group A all defects were fully filled with repair tissue and at 6 months post-surgery most of the repair site was filled with hyaline cartilage. In contrast, in group B all defects were partially filled with repair tissue, but only half of the repair tissue was hyaline cartilage. Defects were only filled with fibrotic tissue in group C. Indeed, histological grading score analysis revealed that an average score in group A was higher than in groups B and C. GAG and type II collagen content and biomechanical property detection showed that the group A levels approached those of normal cartilage. In conclusion, ADSC-loaded cartilage ECM scaffolds induced cartilage repair tissue comparable to native cartilage in terms of mechanical properties and biochemical components.

Electrospun fibre diameter, not alignment, affects mesenchymal stem cell differentiation into the tendon/ligament lineage.

Abstract: Efforts to develop engineered tendons and ligaments have focused on the use of a biomaterial scaffold and a stem cell source. However, the ideal scaffold microenvironment to promote stem cell differentiation and development of organized extracellular matrix is unknown. Through electrospinning, fibre scaffolds can be designed with tailorable architectures to mimic the intended tissue. In this study, the effects of fibre diameter and orientation were examined by electrospinning thin mats, consisting of small ( 2 µm) diameter fibres with either random or aligned fibre orientation. C3H10T1/2 model stem cells were cultured on the six different electrospun mats, as well as smooth spin-coated films, and the morphology, growth and expression of tendon/ligament genes were evaluated. The results demonstrated that fibre diameter affects cellular behaviour more significantly than fibre alignment. Initially, cell density was greater on the small fibre diameter mats, but similar cell densities were found on all mats after an additional week in culture. After 2 weeks, gene expression of collagen 1α1 and decorin was increased on all mats compared to films. Expression of the tendon/ligament transcription factor scleraxis was suppressed on all electrospun mats relative to spin-coated films, but expression on the large-diameter fibre mats was consistently greater than on the medium-diameter fibre mats. These results suggest that larger-diameter fibres (e.g. > 2 µm) may be more suitable for in vitro development of a tendon/ligament tissue. Copyright © 2012 John Wiley & Sons, Ltd.

Initial immune reaction and angiogenesis in bone healing

Abstract: During hematoma formation following injury, an inflammatory reaction ensues as an initial step in the healing process. As granulation tissue matures, revascularization is a prerequisite for successful healing. The hypothesis of this study was that scarless tissue reconstitution in the regenerative bone healing process is dependent on a balanced immune reaction that initiates revasculatory steps. To test this hypothesis, cellular composition and expression profiles of a bone hematoma (regenerative, scarless) was compared with a muscle soft tissue hematoma (healing with a scar) in a sheep model. Upregulation of regulatory T helper cells and anti-inflammatory cytokine expression (IL-10) coincided with an upregulation of angiogenic factors (HIF1α and HIF1α regulated genes) in the regenerative bone hematoma but not in the soft tissue hematoma. These results indicate that the timely termination of inflammation and early onset of revascularization are interdependent and essential for a regenerative healing process. Prolonged pro-inflammatory signaling occurring in a delayed bone-healing model supports the finding that timely termination of inflammation furthers the regenerative process. Differing cellular compositions are due to different cell sources invading the hematoma, determining the ensuing cytokine expression profile and thus paving the path for regenerative healing in bone or the formation of scar tissue in muscle injury.

Fabrication of porous scaffolds by three-dimensional plotting of a pasty calcium phosphate bone cement under mild conditions

Abstract: The major advantage of hydroxyapatite (HA)-forming calcium phosphate cements (CPCs) used as bone replacement materials is their setting under physiological conditions without the necessity for thermal treatment that allows the incorporation of biological factors. In the present study, we have combined the biocompatible consolidation of CPCs with the potential of rapid prototyping (RP) techniques to generate calcium phosphate-based scaffolds with defined inner and outer morphology. We demonstrate the application of the RP technique three-dimensional (3D) plotting for the fabrication of HA cement scaffolds. This was realized by utilizing a paste-like CPC (P-CPC) which is stable as a malleable paste and whose setting reaction is initiated only after contact with aqueous solutions. The P-CPC showed good processability in the 3D plotting process and allowed the fabrication of stable 3D structures of different geometries with adequate mechanical stability and compressive strength. The cytocompatibility of the plotted P-CPC scaffolds was demonstrated in a cell culture experiment with human mesenchymal stem cells. The mild conditions during 3D plotting and post-processing and the realization of the whole procedure under sterile conditions make this approach highly attractive for fabrication of individualized implants with respect to patient-specific requirements by simultaneous plotting of biological components. Copyright © 2012 John Wiley & Sons, Ltd.

Controlled release of BMP-2 from a sintered polymer scaffold enhances bone repair in a mouse calvarial defect model.

Abstract: Sustained and controlled delivery of growth factors, such as bone morphogenetic protein 2 (BMP-2), from polymer scaffolds has excellent potential for enhancing bone regeneration. The present study investigated the use of novel sintered polymer scaffolds prepared using temperature-sensitive PLGA/PEG particles. Growth factors can be incorporated into these scaffolds by mixing the reconstituted growth factor with the particles prior to sintering. The ability of the PLGA/PEG scaffolds to deliver BMP-2 in a controlled and sustained manner was assessed and the osteogenic potential of these scaffolds was determined in a mouse calvarial defect model. BMP-2 was released from the scaffolds in vitro over 3 weeks. On average, ca. 70% of the BMP-2 loaded into the scaffolds was released by the end of this time period. The released BMP-2 was shown to be active and to induce osteogenesis when used in a cell culture assay. A substantial increase in new bone volume of 55% was observed in a mouse calvarial defect model for BMP-2-loaded PLGA/PEG scaffolds compared to empty defect controls. An increase in new bone volume of 31% was observed for PLGA/PEG scaffolds without BMP-2, compared to empty defect controls. These results demonstrate the potential of novel PLGA/PEG scaffolds for sustained BMP-2 delivery for bone-regeneration applications. Copyright © 2012 John Wiley & Sons, Ltd.

Enzymatic mineralization of gellan gum hydrogel for bone tissue-engineering applications and its enhancement by polydopamine.

Abstract: Interest is growing in the use of hydrogels as bone tissue-engineering (TE) scaffolds due to advantages such as injectability and ease of incorporation of active substances such as enzymes. Hydrogels consisting of gellan gum (GG), an inexpensive calcium-crosslinkable polysaccharide, have been applied in cartilage TE. To improve GG suitability as a material for bone TE, alkaline phosphatase (ALP), an enzyme involved in mineralization of bone by cleaving phosphate from organic phosphate, was incorporated into GG hydrogels to induce mineralization with calcium phosphate (CaP). Incorporated ALP induced formation of apatite-like material on the submicron scale within GG gels, as shown by FTIR, SEM, EDS, XRD, ICP-OES, TGA and von Kossa staining. Increasing ALP concentration increased amounts of CaP as well as stiffness. Mineralized GG was able to withstand sterilization by autoclaving, although stiffness decreased. In addition, mineralizability and stiffness of GG was enhanced by the incorporation of polydopamine (PDA). Furthermore, mineralization of GG led to enhanced attachment and vitality of cells in vitro while cytocompatibility of the mineralized gels was comparable to one of the most commonly used bone substitute materials. The results proved that ALP-mediated enzymatic mineralization of GG could be enhanced by functionalization with PDA.

Development of functional biomaterials with micro- and nanoscale technologies for tissue engineering and drug delivery applications

Abstract: Micro- and nanotechnologies have emerged as potentially effective fabrication tools for addressing the challenges faced in tissue engineering and drug delivery. The ability to control and manipulate polymeric biomaterials at the micron and nanometre scale with these fabrication techniques has allowed for the creation of controlled cellular environments, engineering of functional tissues and development of better drug delivery systems. In tissue engineering, micro- and nanotechnologies have enabled the recapitulation of the micro- and nanoscale detail of the cell's environment through controlling the surface chemistry and topography of materials, generating 3D cellular scaffolds and regulating cell-cell interactions. Furthermore, these technologies have led to advances in high-throughput screening (HTS), enabling rapid and efficient discovery of a library of materials and screening of drugs that induce cell-specific responses. In drug delivery, controlling the size and geometry of drug carriers with micro- and nanotechnologies have allowed for the modulation of parametres such as bioavailability, pharmacodynamics and cell-specific targeting. In this review, we introduce recent developments in micro- and nanoscale engineering of polymeric biomaterials, with an emphasis on lithographic techniques, and present an overview of their applications in tissue engineering, HTS and drug delivery.

Improved expansion of human bone marrow-derived mesenchymal stem cells in microcarrier-based suspension culture.

Abstract: Human bone marrow-derived mesenchymal stem cells (hBM-MSCs) have potential clinical utility in the treatment of a multitude of ailments and diseases, due to their relative ease of isolation from patients and their capacity to form many cell types. However, hBM-MSCs are sparse, and can only be isolated in very small quantities, thereby hindering the development of clinical therapies. The use of microcarrier-based stirred suspension bioreactors to expand stem cell populations offers an approach to overcome this problem. Starting with standard culture protocols commonly reported in the literature, we have successfully developed new protocols that allow for improved expansion of hBM-MSCs in stirred suspension bioreactors using CultiSpher-S microcarriers. Cell attachment was facilitated by using intermittent bioreactor agitation, removing fetal bovine serum, modifying the stirring speed and manipulating the medium pH. By manipulating these parameters, we enhanced the cell attachment efficiency in the first 8 h post-inoculation from 18% (standard protocol) to 72% (improved protocol). Following microcarrier attachment, agitation rate was found to impact cell growth kinetics, whereas feeding had no significant effect. By serially subculturing hBM-MSCs using the new suspension bioreactor protocols, we managed to obtain cell fold increases of 10³ within 30 days, which was superior to the 200-fold increase obtained using the standard protocol. The cells were found to retain their defining characteristics after several passages in suspension. This new bioprocess represents a more efficient approach for generating large numbers of hBM-MSCs in culture, which in turn should facilitate the development of new stem cell-based therapies.

Explant culture: a simple, reproducible, efficient and economic technique for isolation of mesenchymal stromal cells from human adipose tissue and lipoaspirate

Abstract: Adipose tissue has emerged as a preferred source of mesenchymal stem/stromal cells (MSC), due to its easy accessibility and high MSC content. The conventional method of isolation of adipose tissue-derived stromal cells (ASC) involves enzymatic digestion and centrifugation, which is a costly and time-consuming process. Mechanical stress during isolation, use of bacterial-derived products and potential contamination with endotoxins and xenoantigens are other disadvantages of this method. In this study, we propose explant culture as a simple and efficient process to isolate ASC from human adipose tissue. This technique can be used to reproducibly isolate ASC from fat tissue obtained by liposuction as well as surgical resection, and yields an enriched ASC population free from contaminating haematopoietic cells. We show that explanting adipose tissue results in a substantially higher yield of ASC at P0 per gram of initial fat tissue processed, as compared to that obtained by enzymatic digestion. We demonstrate that ASC isolated by explant culture are phenotypically and functionally equivalent to those obtained by enzymatic digestion. Further, the explant-derived ASC share the immune privileged status and immunosuppressive properties implicit to MSC, suggesting that they are competent to be tested and applied in allogeneic clinical settings. As explant culture is a simple, inexpensive and gentle method, it may be preferred over the enzymatic technique for obtaining adipose tissue-derived stem/stromal cells for tissue engineering and regenerative medicine, especially in cases of limited starting material.

Glycosaminoglycans enhance osteoblast differentiation of bone marrow derived human mesenchymal stem cells

Abstract: Extracellular matrix plays an important role in regulating cell growth and differentiation. The biomimetic approach of cell-based tissue engineering is based on mirroring this in vivo micro environment for developing a functional tissue engineered construct. In this study, we treated normal tissue culture plates with selected extracellular matrix components consisting of glycosaminoglycans such as chondroitin-4-sulphate, dermatan sulphate, chondroitin-6-sulphate, heparin and hyaluronic acid. Mesenchymal stem cells isolated from adult human bone marrow were cultured on the glycosaminoglycan treated culture plates to evaluate their regulatory role in cell growth and osteoblast differentiation. Although no significant improvement on human mesenchymal stem cell adhesion and proliferation was observed on the glycosaminoglycan-treated tissue culture plates, there was selective osteoblast differentiation, indicating its potential role in differentiation rather than proliferation. Osteoblast differentiation studies showed high osteogenic potential for all tested glycosaminoglycans except chondroitin-4-sulphate. Osteoblast differentiation-associated genes such as osterix, osteocalcin, integrin binding sialoprotein, osteonectin and collagen, type 1, alpha 1 showed significant upregulation. We identified osterix as the key transcription factor responsible for the enhanced bone matrix deposition observed on hyaluronic acid, heparin and chondroitin-6-sulphate. Hyaluronic acid provided the most favourable condition for osteoblast differentiation and bone matrix synthesis. Our results confirm and emphasise the significant role of extracellular matrix in regulating cell differentiation. To summarise, glycosaminoglycans of extracellular matrix played a significant role in regulating osteoblast differentiation and could be exploited in the biomimetic approach of fabricating or functionalizing scaffolds for stem cell based bone tissue engineering.

Successful recellularization of human tendon scaffolds using adipose-derived mesenchymal stem cells and collagen gel.

Abstract: The major goal of regenerative medicine is to determine experimental techniques that take maximal advantage of reparative processes that occur naturally in the animal body. Injection of mesenchymal stem cells into the core of a damaged tendon represents such an approach. Decellularization of native tendons as potential targets and seeding protocols are currently under investigation. The aim of our study was to manufacture a recellularized biocompatible scaffold from cadaveric tissue for use in total or partial tendon injuries. Results showed that it was possible to introduce proliferating cells into the core of a decellularized tendon to treat the scaffold with a collagen gel. The method was effective in maintaining scaffold extracellular matrix and for expressing collagen type I and cartilage oligomeric matrix protein by injecting mesenchymal stem cells.

Evaluation of a novel collagen/gelatin scaffold for achieving the sustained release of basic fibroblast growth factor in a diabetic mouse model

Abstract: The objective of this study was to evaluate the ability of a scaffold, collagen-gelatin sponge (CGS), to release basic fibroblast growth factor (bFGF) in a sustained manner, using a pressure-induced decubitus ulcer model involving genetically diabetic mice. We confirmed that CGSs impregnated with a bFGF concentration of up to 50 µg/cm(2) were able to sustain the release of bFGF throughout their biodegradation. We prepared decubitus ulcers on diabetic mice. After debriding the ulcers, we implanted CGSs (diameter 8 mm) impregnated with normal saline solution (NSS) or bFGF solution (7, 14, 28 or 50 µg/cm(2)). At 1 and 2 weeks after implantation, the mice were sacrificed and tissue specimens were obtained. The wound area, neoepithelium length and numbers and total area of newly formed capillaries were evaluated. The CGSs impregnated with NSS became infected and degraded, whereas the CGSs impregnated with 7 or 14 µg/cm(2) bFGF displayed accelerated dermis-like tissue formation and the CGSs impregnated with 14 µg/cm(2) bFGF produced significant improvements in the remaining wound area, neoepithelium length and numbers and total area of newly formed capillaries compared with the NSS group. No significant difference was observed between the NSS and 50 µg/cm(2) bFGF groups. CGSs impregnated with 7-14 µg/cm(2) bFGF accelerated wound healing, and an excess amount of bFGF did not increase the wound-healing efficacy of the CGSs. Our CGS is a scaffold that can release positively charged growth factors such as bFGF in a sustained manner and shows promise as a scaffold for skin regeneration.

Nanofibrous nerve conduit-enhanced peripheral nerve regeneration.

Abstract: Fibre structures represent a potential class of materials for the formation of synthetic nerve conduits due to their biomimicking architecture. Although the advantages of fibres in enhancing nerve regeneration have been demonstrated, in vivo evaluation of fibre size effect on nerve regeneration remains limited. In this study, we analyzed the effects of fibre diameter of electrospun conduits on peripheral nerve regeneration across a 15-mm critical defect gap in a rat sciatic nerve injury model. By using an electrospinning technique, fibrous conduits comprised of aligned electrospun poly (e-caprolactone) (PCL) microfibers (981 ± 83 nm, Microfiber) or nanofibers (251 ± 32 nm, Nanofiber) were obtained. At three months post implantation, axons regenerated across the defect gap in all animals that received fibrous conduits. In contrast, complete nerve regeneration was not observed in the control group that received empty, non-porous PCL film conduits (Film). Nanofiber conduits resulted in significantly higher total number of myelinated axons and thicker myelin sheaths compared to Microfiber and Film conduits. Retrograde labeling revealed a significant increase in number of regenerated dorsal root ganglion sensory neurons in the presence of Nanofiber conduits (1.93 ± 0.71 × 103 vs. 0.98 ± 0.30 × 103 in Microfiber, p < 0.01). In addition, the compound muscle action potential (CMAP) amplitudes were higher and distal motor latency values were lower in the Nanofiber conduit group compared to the Microfiber group. This study demonstrated the impact of fibre size on peripheral nerve regeneration. These results could provide useful insights for future nerve guide designs. Copyright © 2012 John Wiley & Sons, Ltd.

Chemical surface modification of poly-ε-caprolactone improves Schwann cell proliferation for peripheral nerve repair.

Abstract: Poly-e-caprolactone (PCL) is a biodegradable and biocompatible polymer used in tissue engineering for various clinical applications. Schwann cells (SCs) play an important role in nerve regeneration and repair. SCs attach and proliferate on PCL films but cellular responses are weak due to the hydrophobicity and neutrality of PCL. In this study, PCL films were hydrolysed and aminolysed to modify the surface with different functional groups and improve hydrophilicity. Hydrolysed films showed a significant increase in hydrophilicity while maintaining surface topography. A significant decrease in mechanical properties was also observed in the case of aminolysis. In vitro tests with Schwann cells (SCs) were performed to assess film biocompatibility. A short-time experiment showed improved cell attachment on modified films, in particular when amino groups were present on the material surface. Cell proliferation significantly increased when both treatments were performed, indicating that surface treatments are necessary for SC response. It was also demonstrated that cell morphology was influenced by physico-chemical surface properties. PCL can be used to make artificial conduits and chemical modification of the inner lumen improves biocompatibility.

Periodontal regeneration using an injectable bone cement combined with BMP-2 or FGF-2

Abstract: Periodontitis is a frequently diagnosed oral disease characterized by bone resorption and soft tissue loss around teeth. Unfortunately, currently available therapies only slow or arrest progress of the disease. Ideally, treatment of periodontal defects should be focused on complete regeneration of the lost tissues [(bone and periodontal ligament (PDL)]. As a result, this study used intrabony defects to evaluate the regenerative potential of an injectable macroporous calcium phosphate cement (CaP) in combination with bone morphogenetic protein-2 (BMP-2) or fibroblast growth factor-2 (FGF-2). After creating 30 periodontal defects in 15 Wistar rats, three treatment strategies were conducted: application of CaP only, CaP + BMP-2 and CaP + FGF-2. Animals were euthanized after 12 weeks and processed for histology and histomorphometry. Using CaP alone resulted in limited effects on PDL and bone healing. CaP + BMP-2 showed a good response for bone healing; a significant 2.4 fold increase in bone healing score was observed compared to CaP. However, for PDL healing, CaP + BMP-2 treatment showed no difference compared to the CaP group. The best results were observed with the combined treatment of CaP + FGF-2, which showed a significant 3.3 fold increase in PDL healing score compared to CaP + BMP-2 and a significant 2.6 fold increase compared to CaP. For bone healing, CaP + FGF-2 showed a significant 1.9 fold increase compared to CaP but no significant difference was noted compared to the CaP + BMP-2 group. The combination of a topical application of FGF-2 and an injectable CaP seems to be a promising treatment modality for periodontal regeneration.

Induction of bone formation in biphasic calcium phosphate scaffolds by bone morphogenetic protein-2 and primary osteoblasts

Abstract: Bone tissue engineering strategies mainly depend on porous scaffold materials. In this study, novel biphasic calcium phosphate (BCP) matrices were generated by 3D-printing. High porosity was achieved by starch consolidation. This study aimed to characterise the porous BCP-scaffold properties and interactions of osteogenic cells and growth factors under in vivo conditions. Five differently treated constructs were implanted subcutaneously in syngeneic rats: plain BCP constructs (group A), constructs pre-treated with BMP-2 (group B; 1.6 µg BMP-2 per scaffold), seeded with primary osteoblasts (OB) (group C), seeded with OB and BMP-2 (group D) and constructs seeded with OB and pre-cultivated in a flow bioreactor for 6 weeks (group E). After 2, 4 and 6 weeks, specimens were explanted and subjected to histological and molecular biological analyses. Explanted scaffolds were invaded by fibrovascular tissue without significant foreign body reactions. Morphometric analysis demonstrated significantly increased bone formation in samples from group D (OB + BMP-2) compared to all other groups. Samples from groups B-E displayed significant mRNA expression of bone-specific genes after 6 weeks. Pre-cultivation in the flow bioreactor (group E) induced bone formation comparable with group B. In this study, differences in bone distribution between samples with BMP-2 or osteoblasts could be observed. In conclusion, combination of osteoblasts and BMP-2 synergistically enhanced bone formation in novel ceramic scaffolds. These results provide the basis for further experiments in orthotopic defect models with a focus on future applications in orthopaedic and reconstructive surgery. Copyright © 2012 John Wiley & Sons, Ltd.

Promotion of diabetic wound healing by collagen scaffold with collagen‐binding vascular endothelial growth factor in a diabetic rat model

Abstract: Reference EPFL-CONF-201377View record in Web of Science Record created on 2014-08-29, modified on 2017-05-12

Shear stress influences the pluripotency of murine embryonic stem cells in stirred suspension bioreactors.

Abstract: Pluripotent embryonic stem cells (ESCs) have been used increasingly in research as primary material for various tissue-engineering applications. Pluripotency, or the ability to give rise to all cells of the body, is an important characteristic of ESCs. Traditional methods use leukaemia inhibitory factor (LIF) to maintain murine embryonic stem cell (mESC) pluripotency in static and bioreactor cultures. When LIF is removed from mESCs in static cultures, pluripotency genes are downregulated and the cultures will spontaneously differentiate. Recently we have shown the maintenance of pluripotency gene expression of mESCs in stirred suspension bioreactors during differentiation experiments in the absence of LIF. This is undesired in a differentiation experiment, where the goal is downregulation of pluripotency gene expression and upregulation of gene expression characteristic to the differentiation. Thus, the objective of this study was to examine how effectively different levels of shear stress [100 rpm (6 dyne/cm2), 60 rpm (3 dyne/cm2)] maintained and influenced pluripotency in suspension bioreactors. The pluripotency markers Oct-4, Nanog, Sox-2 and Rex-1 were assessed using gene expression profiles and flow-cytometry analysis and showed that shear stress does maintain and influence the gene expression of certain pluripotency markers. Some significant differences between the two levels of shear stress were seen and the combination of shear stress and LIF was observed to synergistically increase the expression of certain pluripotency markers. Overall, this study provides a better understanding of the environmental conditions within suspension bioreactors and how these conditions affect the pluripotency of mESCs. Copyright © 2012 John Wiley & Sons, Ltd.

Design, fabrication and in vitro evaluation of a novel polymer‐hydrogel hybrid scaffold for bone tissue engineering

Abstract: The development of a bone mechanically-compatible and osteoinductive scaffold is important for bone tissue engineering applications, particularly for the repair and regeneration of large area critically-sized bone defects. Although previous studies with weight-bearing scaffolds have shown promising results, there is a clear need to develop better osteoinductive strategies for effective scaffold-based bone regeneration. In this study, we designed and fabricated a novel polymer-hydrogel hybrid scaffold system in which a load-bearing polymer matrix and a peptide hydrogel allowed for the synergistic combination of mechanical strength and great potential for osteoinductivity in a single scaffold. The hybrid scaffold system promoted increased pre-osteoblastic cell proliferation. Further, we biotinylated human recombinant bone morphogenetic protein 2 (rhBMP2), and characterized the biotin addition and its effect on rhBMP2 biological activity. The biotinylated rhBMP2 was tethered to the hybrid scaffold using biotin-streptavidin complexation. Controlled release studies demonstrated increased rhBMP2 retention with the tethered rhBMP2 hybrid scaffold group. In vitro evaluation of the hybrid scaffold was performed with rat bone marrow stromal cells and mouse pre-osteoblast cell line MC3T3-E1 cells. Gene expression of alkaline phosphatase (ALP), collagen I (Col I), osteopontin (OPN), bone sialoprotein (BSP), Runx-2 and osteocalcin (OC) increased in MC3T3-E1 cells seeded on the rhBMP2 tethered hybrid scaffolds over the untethered counterparts, demonstrating osteoinductive potential of the hybrid graft. These findings suggest the possibility of developing a novel polymer-hydrogel hybrid system that is weight bearing and osteoinductive for effective bone tissue engineering.

Enhancing human nucleus pulposus cells for biological treatment approaches of degenerative intervertebral disc diseases: a systematic review.

Abstract: Intervertebral disc (IVD) degeneration has been described as an aberrant, cell-mediated, age- and genetics-dependent molecular degeneration process, which can be accelerated by nutritional, mechanical and toxic factors. Collective involvement of these factors can result in structural failures, which are often associated with pain. Current treatment approaches are restricted to symptomatic therapies, not addressing options of restoring structural or biological deterioration of the IVD as the underlying problem. Therapeutic potentials of IVD cell transplantation, biomaterials, inhibiting or activating bioactive factors, including gene-therapeutic approaches, have been shown in vitro or in small animal models. Since human degenerative IVD cells display distinctive features with regard to cell biology and regenerative potential, we attempted a systematic review, investigating the in vitro response of human nucleus pulposus cells to different stimuli. Therefore, we conducted an electronic database search on Medline through July 2011 to identify, compare and discuss publications concerning the effects of cell-cell stimulation, bioactive factors, biomaterials and combinations thereof in terms of cell isolation, proliferation, differentiation and matrix protein synthesis. This survey and discussion might serve as a source for designing future biological treatment strategies for the human IVD.

Three‐dimensional growth matrix for human embryonic stem cell‐derived neuronal cells

Abstract: The future of tissue engineering applications for neuronal cells will require a supportive 3D matrix. This particular matrix should be soft, elastic and supportive for cell growth. In this study, we characterized the suitability of a 3D synthetic hydrogel matrix, PuraMatrix™, as a growth platform for human embryonic stem cell (hESC)-derived neural cells. The viability of the cells grown on top of, inside and under the hydrogel was monitored. The maturation and electrical activity of the neuronal networks inside the hydrogel were further characterized. We showed that cells stayed viable on the top of the PuraMatrix™ surface and growth of the neural cells and neural processes was good. Further, hESC-derived neurons, astrocytes and oligodendrocytes all grew, matured and migrated when cultured inside the hydrogel. Importantly, neuronal cells were able to form electrically active connections that were verified using microelectrode array. Thus, PuraMatrix is a good supportive growth matrix for human neural cells and may serve as a matrix for neuronal scaffolds in neural tissue engineering. Copyright © 2012 John Wiley & Sons, Ltd.

Development and evaluation of axially aligned nanofibres for blood vessel tissue engineering

Abstract: Biodegradable polymers have been extensively used as scaffolds to regenerate lost tissues. The geometry of the three-dimensional (3D) scaffolds has an influence on the cellular behaviour. In this study, we have developed 3D-scaffolds of axially aligned nanofibres of poly(lactic acid) (PLA), poly(caprolactone) (PCL) and PLA:CL (50:50) with diameters in the range 100–400 nm, internal diameter 4 mm, length 4 cm and wall thickness 0.2 mm, by using a dynamic collector. PCL and PLA:CL nanofibres were significantly less hydrophobic than PLA nanofibres. The porosity of PCL (16.23 ± 9.88%) and PLA:CL nanofibres (14.77 ± 3.41%) were comparable, while PLA (6.57 ± 1.54%) nanofibres had lower porosity. The tensile strength and Young's modulus of PLA was significantly lower than PCL and PLA:CL nanofibres and the suture retention strengths of all three scaffolds were comparable. After 4 weeks, the molecular weight of PLA nanofibres was reduced by 53% compared to 44% and 41% for PCL and the PLA:CL nanofibres, respectively. However, the PLA:CL nanofibres maintained their structural integrity even after 28 days. Platelet adhesion studies showed that PCL nanofibres had least tendency to be thrombogenic, while PLA:CL blend nanofibres were highly thrombogenic. Further, in vitro responses such as cell adhesion, proliferation and gene expression of human umbilical vascular endothelial cells (HUVECs) were evaluated. After 6 days of culture, the surfaces of all the three scaffolds were completely covered with cells. Our results demonstrate that expression levels of elastin, angiopoietin, laminin-4α and -5α were upregulated in PCL and PLA:CL nanofibres without the addition of any exogenous factors. Copyright © 2012 John Wiley & Sons, Ltd.

Recombinant fibroblast growth protein enhances healing ability of experimentally induced tendon injury in vivo.

Abstract: This study was designed to investigate the effects of recombinant human basic fibroblast growth factor (bFGF) on a complete superficial digital flexor tendon (SDFT) rupture after surgical repair in rabbits. Eighty mature New Zealand White rabbits of both sexes were randomly divided into two equal groups: Treated and Control. Each group was subdivided into two 28- and 84-day post-injury subgroups. After tenotomy and surgical repair, the animals were immobilized for 14 days. In the treated group, bFGF was directly applied subcutaneously over the lesion on days 3, 7 and 10 after injury. The control animals received normal saline injection of the same viscosity and volume and at the same intervals. Ultrasonographical observations were conducted at weekly intervals. The animals were euthanized at 28 and 84 days after injury. The tendons were evaluated at macroscopic, histopathologic and ultrastructural levels and were assessed for biomechanical and percentage dry weight parameters. Compared to injured control animals, treated animals showed a decrease in the diameter of the injured tendon and peritendinous adhesion as well as increased tenoblast proliferation, collagen production and ultimate strength of the injured tendons (p < 0.005). At 84 days after injury, treatment resulted in enhanced maturation of the cellular and collagen elements and improved tissue alignment and density. These improvements resulted in increased biomechanical performance of the injured tendons compared to controls (p = 0.001). bFGF showed promising curative effects on restoration of the biomechanical and morphological properties of the ruptured SDFT in rabbits and may be applicable in clinical studies.

The interaction between β1 integrins and ERK1/2 in osteogenic differentiation of human mesenchymal stem cells under fluid shear stress modelled by a perfusion system.

Abstract: Fluid shear stress (FSS) is an important biomechanical factor regulating the osteogenic differentiation of human mesenchymal stem cells (hMSCs) and is therefore widely used in bone tissue engineering. However, the mechanotransduction of FSS in hMSCs remains largely unknown. As β1 integrins are considered to be important mechanoreceptors in other cells, we suspect that β1 integrins should also be important for hMSCs to sense the stimulation of FSS. We used a perfusion culture system to produce FSS loading on hMSCs seeded in PLGA three-dimensional (3D) scaffolds and investigated the roles of β1 integrins, FAK and ERK1/2 in FSS-induced osteogenic differentiation of hMSCs. Our results showed that FSS not only markedly increased ALP activity and the expression of ALP, OCN, Runx2 and COLIα genes but also significantly enhanced the phosphorylation of ERK1/2, Runx2 and FAK. FSS-induced activation of ERK1/2 and FAK was inhibited by blockade of the connection between β1 integrins and ECM with RGDS peptide and integrins β1 monoclonal antibody. Our study also found that FSS could upregulate the expression level of β1 integrins and that this upregulation could be abolished by PD98059. Further investigation indicated that FSS-activated ERK1/2 led to the phosphorylation of IκBα and NFκB p65. The activation of NFκB p65 resulted in the upregulation of β1 integrin expression. Therefore, it could be inferred that β1 integrins should sense the stimulation of FSS and thus activate ERK1/2 through activating of FAK, and FSS-activated ERK1/2 feedback to upregulate the expression of β1 integrins through activating NFκB.

Enhanced endothelial differentiation of adipose-derived stem cells by substrate nanotopography.

Abstract: Adipose-derived stem cells (ADSCs) have great potential as a cell source for tissue engineering and regenerative medicine because they are easier to obtain, have lower donor-site morbidity and are available in larger numbers than stem cells harvested using bone marrow aspiration. Until now, little has been known about how nanotopography affects the proliferation and endothelial differentiation of ADSCs. In the present study, two nanograting substrates with a period (ridge and groove) of about 250 and 500 nm, respectively, were fabricated on quartz and their effect on ADSC fate was investigated. The results showed that proliferation of ADSCs on nanograting substrates decreased while cell attachment was not significantly affected compared to a flat substrate. Endothelial differentiation of ADSCs on both flat and nanograting substrates can be induced with vascular endothelial growth factor, as shown by immunofluorescent staining. Quantitative real-time PCR analysis showed significantly enhanced upregulation of vWF, PECAM-1 and VE-cadherin at the gene level by ADSCs on the nanograting substrates. In vitro angiogenesis assay on Matrigel showed that nanograting substrates enhanced capillary tube formation. This study highlights the beneficial influence of nanotopography on the differentiation of ADSC into endothelial cells which play an important role in vascularization.

Gene delivery of bone morphogenetic protein-2 plasmid DNA promotes bone formation in a large animal model.

Abstract: In the field of bone regeneration, BMP-2 is considered one of the most important growth factors because of its strong osteogenic activity, and is therefore extensively used in clinical practice. However, the short half-life of BMP-2 protein necessitates the use of supraphysiological doses, leading to severe side-effects. This study investigated the efficiency of bone formation at ectopic and orthotopic sites as a result of a low-cost, prolonged presence of BMP-2 in a large animal model. Constructs consisting of alginate hydrogel and BMP-2 cDNA, together acting as a non-viral gene-activated matrix, were combined with goat multipotent stromal cells (gMSCs) and implanted in spinal cassettes or, together with ceramic granules, intramuscularly in goats, both for 16 weeks. Bone formation occurred in all cell-seeded ectopic constructs, but the constructs containing both gMSCs and BMP-2 plasmid DNA showed higher collagen I and bone levels, indicating an osteogenic effect of the BMP-2 plasmid DNA. This was not seen in unseeded constructs, even though transfected, BMP-2-producing cells were detected in all constructs containing plasmid DNA. Orthotopic constructs showed mainly bone formation in the unseeded groups. Besides bone, calcified alginate was present in these groups, acting as a surface for new bone formation. In conclusion, transfection of seeded or resident cells from this DNA delivery system led to stable expression of BMP-2 during 16 weeks, and promoted osteogenic differentiation and subsequent bone formation in cell-seeded constructs at an ectopic location and in cell-free constructs at an orthotopic location in a large animal model.

Characterization of a macroporous polyvinyl alcohol scaffold for the repair of focal articular cartilage defects.

Abstract: Focal cartilage defects reduce the ability of articular cartilage to resist mechanical loading and provide lubrication during joint motion. The limitations in current surgical treatments have motivated the use of biocompatible scaffolds as a future treatment option. Here we describe a second-generation, macroporous, polyvinyl alcohol (PVA) scaffold with independently tunable morphological and mechanical properties. The compressive moduli of the PVA scaffold increased with increasing polymer concentration and applied compressive strain, with values in the range for human articular cartilage (HA >1000 kPa, EY >500 kPa). Scaffolds also possessed strain-dependent permeability and Poisson’s ratio. The interconnected macroporous network was found to facilitate chondrocyte seeding and proliferation through the scaffold over 1 week in culture. Overall, these promising characteristics demonstrate the potential of this macroporous scaffold for future studies in focal cartilage defect repair.

Local injection of lovastatin in biodegradable polyurethane scaffolds enhances bone regeneration in a critical‐sized segmental defect in rat femora

Abstract: Statins, a class of naturally-occurring compounds that inhibit HMG-CoA reductase, are known to increase endogenous bone morphogenetic protein-2 (BMP-2) expression. Local administration of statins has been shown to stimulate fracture repair in in vivo animal experiments. However, the ability of statins to heal more challenging critical-sized defects at the mid-diaphyseal region in long bones has not been investigated. In this study, we examined the potential of injectable lovastatin microparticles combined with biodegradable polyurethane (PUR) scaffolds in preclinical animal models: metaphyseal small plug defects and diaphyseal segmental bone defects in rat femora. Sustained release of lovastatin from the lovastatin microparticles was achieved over 14 days. The released lovastatin was bioactive, as evidenced by its ability to stimulate BMP-2 gene expression in osteoblastic cells. Micro-computed tomography (CT) and histological examinations showed that lovastatin microparticles, injected into PUR scaffolds implanted in femoral plug defects, enhanced new bone formation. Furthermore, bi-weekly multiple injections of lovastatin microparticles into PUR scaffolds implanted in critical-sized femoral segmental defects resulted in increased new bone formation compared to the vehicle control. In addition, bridging of the defect with newly formed bone was observed in four of nine defects in the lovastatin microparticle treatment group, whereas none of the defects in the vehicle group showed bridging. These observations suggest that local delivery of lovastatin combined with PUR scaffold can be an effective approach for treatment of orthopaedic bone defects and that multiple injections of lovastatin may be useful for large defects. Copyright © 2012 John Wiley & Sons, Ltd.

Development and evaluation of a decellularized membrane from human dermis

Abstract: Interest is increasing in biological scaffolds for tissue regeneration, such as extracellular matrix (ECM) membranes, developed through soft tissue decellularization. The present study describes the development of a chemicophysical decellularization method applied to allogenic human-derived dermis (HDM). To evaluate the absence of viable cells and the maintenance of ECM structure, biological, histological and ultrastructural assessments were performed on the HDM membrane. Residual DNA content and glycosaminoglycan (GAG) and collagen contents were quantified. Growth factor (GF) release was directly measured on HDM extracts and indirectly measured by assessing cell proliferation after administering extract to cultures. Tensile tests were performed to measure the effect of the decellularization technique on the mechanical properties of tissue. Histocompatibility was investigated after subcutaneous implantation in rats. Residual DNA, GAG and collagen content measurements, vitality index, histology and electron microscopy showed the efficiency of the decellularization process and preservation of ECM matrix and bioactivity. In HDM extracts, among the tested GFs, transforming growth factor-β1 showed the highest concentration. HDM extracts significantly increased the proliferation rate of L929 fibroblasts in comparison with controls (p < 0.005, p < 0.05 and p < 0.0005). Maximum load and stiffness of HDM were significantly higher than those of cellularized dermis (p < 0.0005, p < 0.005). Histological and histomorphometric analysis of explanted samples showed that the membrane was integrated with host tissues in the absence of inflammatory reactions. Our results show that the decellularization method allowed the development of a human allograft dermal matrix that might be useful for soft tissue regeneration. Copyright © 2012 John Wiley & Sons, Ltd.