Loading of BMP‐2‐related peptide onto three‐dimensional nano‐hydroxyapatite scaffolds accelerates mineralization in critical‐sized cranial bone defects

Extrusion free‐forming, as a rapid prototyping technique, is extensively applied in fabricating ceramic material in bone tissue engineering. To improve the osteoinductivity of nano‐hydroxyapatite (nHA) scaffold fabricated by extrusion free‐forming, in this study, we incorporated a new peptide (P28) and optimized the superficial microstructure after shaping by controlling the sintering temperature. P28, a novel bone morphogenic protein 2 (BMP‐2)‐related peptide, was designed in this study. Analysis of the structure, physicochemical properties and release kinetics of P28 from nHA sintered at temperatures ranging from 1000 °C to 1400 °C revealed that nHA sintered at 1000 °C had higher porosity, preferable pore size and better capacity to control P28 release than that sintered at other temperatures. Moreover, the nHA scaffold sintered at 1000 °C with P28 showed improved adhesion, proliferation and osteogenic differentiation of MC3T3‐E1 cells compared with scaffolds lacking P28 or BMP‐2. In vivo, nHA scaffolds sintered at 1000 °C with P28 or BMP‐2 induced greater bone regeneration in critical‐sized rat cranial defects at 6 and 12 weeks post‐implantation compared with scaffolds lacking P28 or BMP‐2. Thus, nHA scaffolds sintered at 1000 °C and loaded with P28 may be excellent biomaterials for bone tissue engineering. Copyright © 2016 John Wiley & Sons, Ltd.     

Repair of an osteochondral defect by sustained delivery of BMP‐2 or TGFβ1 from a bilayered alginate–PLGA scaffold

Regeneration of cartilage defects can be accelerated by localized delivery of appropriate growth factors (GFs) from scaffolds. In the present study we analysed the in vitro and in vivo release rates and delivery efficacies of transforming growth factor‐β1 (TGFβ1) and bone morphogenetic protein‐2 (BMP‐2) from a bilayered system, applied for osteochondral defect repair in a rabbit model. A bone‐orientated, porous PLGA cylinder was overlaid with GF containing PLGA microspheres, dispersed in an alginate matrix. Four microsphere formulations were incorporated: (a) blank ones; (b) microspheres containing 50 ng TGFβ1; (c) microspheres containing 2.5 µg BMP‐2; and (d) microspheres containing 5 µg BMP‐2. Release kinetics and tissue distributions were determined using iodinated (¹²⁵I) GFs. Bioactivity of in vitro released BMP‐2 and TGFβ1 was confirmed in cell‐based assays. In vivo release profiles indicated good GF release control. 20% of BMP‐2 and 15% of TGFβ1 were released during the first day. Virtually the total dose was delivered at the end of week 6. Significant histological differences were observed between untreated and GF‐treated specimens, there being especially relevant short‐term outcomes with 50 ng TGFβ1 and 5 µg BMP‐2. Although the evaluation scores for the newly formed cartilage did not differ significantly, 5 µg BMP‐2 gave rise to higher quality cartilage with improved surface regularity, tissue integration and increased collagen‐type II and aggrecan immunoreactivity 2 weeks post‐implantation. Hence, the bilayered system controlled GF release rates and led to preserved cartilage integrity from 12 weeks up to at least 24 weeks. 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

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.     

BMP‐2‐transduced human bone marrow stem cells enhance neo‐bone formation in a rat critical‐sized femur defect

Synthetic graft materials are considered as possible substitutes for cancellous bone, but lack osteogenic and osteoinductive properties. In this study, we investigated how composite scaffolds of βTCP containing osteogenic human bone marrow mesenchymal stem cells (hBMSCs) and osteoinductive bone morphogenetic protein‐2 (BMP‐2) influenced the process of fracture healing. hBMSCs were loaded into βTCP scaffolds 24 h before implantation in a rat critical‐sized bone defect. hBMSCs were either stimulated with rhBMP‐2 or transduced with BMP‐2 by gene transfer. The effect of both protein stimulation and gene transfer was compared for osteogenic outcome. X‐rays were conducted at weeks 0, 1, 3, 6, 9 and 12 post‐operatively. In addition, bone‐labelling fluorochromes were applied at 0, 3, 6 and 9 weeks. Histological analysis was performed for the amount of callus tissue and cartilage formation. At 6 weeks, the critical‐sized defect in 33% of the rats treated with the Ad‐BMP‐2‐transduced hBMSCs/βTCP scaffolds was radiographically bridged. In contrast, in only 10% of the rats treated with rhBMP2/hBMSCs, 12 weeks post‐treatment, the bone defect was closed in all treated rats of the Ad‐BMP‐2 group except for one. Histology showed significantly higher amounts of callus formation in both Ad‐BMP‐2‐ and rhBMP‐2‐treated rats. The amount of neocartilage was less pronounced in both BMP‐2‐related groups. In summary, scaffolds with BMP‐2‐transduced hBMSCs performed better than those with the rhBMP2/hBMSCs protein. These results suggest that combinations of osteoconductive biomaterials with genetically modified MSCs capable of secreting osteoinductive proteins may represent a promising alternative for bone regeneration. Copyright © 2015 John Wiley & Sons, Ltd.     

Alveolar bone tissue engineering in critical‐size defects of experimental animal models: a systematic review and meta‐analysis

Regeneration of large, ‘critical‐size’ bone defects remains a clinical challenge. Bone tissue engineering (BTE) is emerging as a promising alternative to autogenous, allogeneic and biomaterial‐based bone grafting. The objective of this systematic review was to answer the focused question: in animal models, do cell‐based BTE strategies enhance regeneration in alveolar bone critical‐size defects (CSDs), compared with grafting with only biomaterial scaffolds or autogenous bone? Following PRISMA guidelines, electronic databases were searched for controlled animal studies reporting maxillary or mandibular CSD and implantation of mesenchymal stem cells (MSCs) or osteoblasts (OBs) seeded on biomaterial scaffolds. A random effects meta‐analysis was performed for the outcome histomorphometric new bone formation (%NBF). Thirty‐six studies were included that reported on large‐ (monkeys, dogs, sheep, minipigs) and small‐animal (rabbits, rats) models. On average, studies presented with an unclear‐to‐high risk of bias and short observation times. In most studies, MSCs or OBs were used in combination with alloplastic mineral‐phase scaffolds. In five studies, cells were modified by ex vivo gene transfer of bone morphogenetic proteins (BMPs). The meta‐analysis indicated statistically significant benefits in favour of: (1) cell‐loaded vs. cell‐free scaffolds [weighted mean difference (WMD) 15.59–49.15% and 8.60–13.85% NBF in large‐ and small‐animal models, respectively]; and (2) BMP‐gene‐modified vs. unmodified cells (WMD 10.06–20.83% NBF in small‐animal models). Results of cell‐loaded scaffolds vs. autogenous bone were inconclusive. Overall, heterogeneity in the meta‐analysis was high (I² > 90%). In summary, alveolar bone regeneration is enhanced by addition of osteogenic cells to biomaterial scaffolds. The direction and estimates of treatment effect are useful to predict therapeutic efficacy and guide future clinical trials of BTE. Copyright © 2016 John Wiley & Sons, Ltd.     

Characterization of recombinant human bone morphogenetic protein‐2 delivery from injectable hyaluronan‐based hydrogels by means of 125I‐radiolabelling

This study presents a thorough in vitro and in vivo characterization of the delivery of bone morphogenetic protein 2 (BMP‐2) from a hyaluronan‐based hydrogel system. The in vitro release of BMP‐2 from similar hydrogels has previously been studied by enzyme‐linked immunosorbent assay (ELISA), by which only a fraction of the loaded protein is detected. In the current study, ¹²⁵I radiolabelling was used instead to monitor BMP‐2 in vitro and in vivo. To minimize protein loss during handling, ¹²⁵I‐BMP‐2 adsorption to different tubes was studied at different times and temperatures. The data showed that Protein LoBind tubes exhibited the lowest protein affinity. Furthermore, a biphasic release profile of biologically active BMP‐2 was observed both in vitro and in vivo, with the initial fast phase during the first week, followed by a slower release during the remaining 3 weeks. The initial fast‐release phase corresponded to the early bone formation observed after 8 days in an ectopic model in rats. Bone volume and mineral content increased until day 14, after which a decrease in bone volume was observed, possibly due to resorption in response to decreased amounts of released BMP‐2. Overall, the results suggested that cautious protein handling and a reliable quantification technique are essential factors for successful design of a BMP‐2 delivery system. Copyright © 2012 John Wiley & Sons, Ltd.     

Osteogenesis of adipose‐derived stem cells on polycaprolactone–β‐tricalcium phosphate scaffold fabricated via selective laser sintering and surface coating with collagen type I

The current study aimed to fabricate three‐dimensional (3D) polycaprolactone (PCL), polycaprolactone and β‐tricalcium phosphate (PCL–TCP) scaffolds via a selective laser‐sintering technique (SLS). Collagen type I was further coated onto PCL–TCP scaffolds to form PCL–TCP–COL scaffolds. The physical characters of these three scaffolds were analysed. The osteogenic potential of porcine adipose‐derived stem cells (pASCs) was compared among these three scaffolds in order to find an optimal scaffold for bone tissue engineering. The experimental results showed no significant differences in pore size and porosity among the three scaffolds; the porosity was ca. 75–77% and the pore size was ca. 300–500 µm in all three. The compressive modulus was increased from 6.77 ± 0.19 to 13.66 ± 0.19 MPa by adding 30% β‐TCP into a 70% PCL scaffold. No significant increase of mechanical strength was found by surface‐coating with collagen type I. Hydrophilicity and swelling ratios showed statistical elevation (p < 0.05) after collagen type I was coated onto the PCL–TCP scaffolds. The in vitro study demonstrated that pASCs had the best osteogenic differentiation on PCL–TCP–COL group scaffolds, due to the highest ALP activity, osteocalcin mRNA expression and mineralization. A nude mice experiment showed better woven bone and vascular tissue formation in the PCL–TCP–COL group than in the PCL group. In conclusion, the study demonstrated the ability to fabricate 3D, porous PCL–TCP composite scaffolds (PCL:TCP = 70:30 by weight) via an in‐house‐built SLS technique. In addition, the osteogenic ability of pASCs was found to be enhanced by coating COL onto the PCL–TCP scaffolds, both in vitro and in vivo. Copyright © 2013 John Wiley & Sons, Ltd.     

Self‐expandable tubular collagen implants

Collagen has been extensively used as a biomaterial, yet for tubular organ repair, synthetic polymers or metals (e.g., stents) are typically used. In this study, we report a novel type of tubular implant solely consisting of type I collagen, suitable to self‐expand in case of minimal invasive implantation. Potential benefits of this collagen scaffold over conventional materials include improved endothelialization, biodegradation over time, and possibilities to add bioactive components to the scaffold, such as anticoagulants. Implants were prepared by compression of porous scaffolds consisting of fibrillar type I collagen (1.0–2.0% (w/v)). By applying carbodiimide cross‐linking to the compressed scaffolds in their opened position, entropy‐driven shape memory was induced. The scaffolds were subsequently crimped and dried around a guidewire. Upon exposure to water, crimped scaffolds deployed within 15–60 s (depending on the collagen concentration used), thereby returning to the original opened form. The scaffolds were cytocompatible as assessed by cell culture with human primary vascular endothelial and smooth muscle cells. Compression force required to compress the open scaffolds increased with collagen content from 16 to 32 mN for 1.0% to 2.0% (w/v) collagen scaffolds. In conclusion, we report the first self‐expandable tubular implant consisting of solely type I collagen that may have potential as a biological vascular implant.     

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

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.     

Improved calvarial bone repair by hASCs engineered with Cre/loxP‐based baculovirus conferring prolonged BMP‐2 and MiR‐148b co‐expression

Repairing large calvarial bone defects remains a challenging task. Previously, it was discovered that that miR‐148b, when acting in concert with bone morphogenetic protein 2 (BMP‐2), enhanced the osteogenesis of human adipose‐derived stem cells (hASCs) and improved calvarial bone healing in nude mice. However, the molecular target of miR‐148b remained elusive. Here it is revealed that miR‐148b directly targets NOG, whose gene product (noggin) is an antagonist to BMPs and negatively regulates BMP‐induced osteogenic differentiation and bone formation. A new Cre/loxP‐based baculovirus system was employed to drive prolonged BMP‐2 and miR‐148b overexpression in hASCs, wherein the BMP‐2 overexpression induced noggin expression but the concurrent miR‐148b expression downregulated noggin, thus relieving the negative regulatory loop and ameliorating hASC osteogenesis without hindering hASC proliferation or triggering appreciable cytotoxicity. Implantation of the engineered hASCs coexpressing BMP‐2 and miR‐148b into nude mice enabled substantial repair of critical‐size calvarial bone defects (4 mm diameter) at 12 weeks post‐transplantation, filling 83% of the defect area, 75% of bone volume and restoring the bone density to 89% of the original bone density. Such superior healing effects indicate the potential of the Cre/loxP‐based baculovirus‐mediated BMP‐2/miR‐148b expression for calvarial bone repair. Copyright © 2016 John Wiley & Sons, Ltd.     

Ultrasound‐responsive gene‐activated matrices for osteogenic gene therapy using matrix‐assisted sonoporation

Gene‐activated matrix (GAM)‐based therapeutics for tissue regeneration are limited by efficacy, the lack of spatiotemporal control and availability of target cells, all of which impact negatively on their translation to the clinic. Here, an advanced ultrasound‐responsive GAM is described containing target cells that facilitates matrix‐assisted sonoporation (MAS) to induce osteogenic differentiation. Ultrasound‐responsive GAMs consisting of fibrin/collagen hybrid‐matrices containing microbubbles, bone morphogenetic protein BMP2/7 coexpression plasmids together with C2C12 cells were treated with ultrasound either in vitro or following parenteral intramuscular implantation in vivo. Using direct measurement for alkaline phosphatase activity, von Kossa staining and immunohistochemical analysis for osteocalcin expression, MAS‐stimulated osteogenic differentiation was confirmed in the GAMs in vitro 7 days after treatment with ultrasound. At day 30 post‐treatment with ultrasound, ectopic osteogenic differentiation was confirmed in vivo using X‐ray microcomputed tomography and histological analysis. Osteogenic differentiation was indicated by the presence of ectopic bone structures in all animals treated with MAS. In addition, bone volumes in this group were statistically greater than those in the control groups. This novel approach of incorporating a MAS capability into GAMs could be exploited to facilitate ex vivo gene transfer with subsequent surgical implantation or alternatively provide a minimally invasive means of stimulating in situ transgene delivery for osteoinductive gene‐based therapies. Copyright © 2017 John Wiley & Sons, Ltd.     

Human tissue‐engineered bone produced in clinically relevant amounts using a semi‐automated perfusion bioreactor system: a preliminary study

The aim of this study was to evaluate a semi‐automated perfusion bioreactor system for the production of clinically relevant amounts of human tissue‐engineered bone. Human bone marrow stromal cells (hBMSCs) of eight donors were dynamically seeded and proliferated in a perfusion bioreactor system in clinically relevant volumes (10 cm³) of macroporous biphasic calcium phosphate scaffolds (BCP particles, 2–6 mm). Cell load and distribution were shown using methylene blue staining. MTT staining was used to demonstrate viability of the present cells. After 20 days of cultivation, the particles were covered with a homogeneous layer of viable cells. Online oxygen measurements confirmed the proliferation of hBMSCs in the bioreactor. After 20 days of cultivation, the hybrid constructs became interconnected and a dense layer of extracellular matrix was present, as visualized by scanning electron microscopy (SEM). Furthermore, the hBMSCs showed differentiation towards the osteogenic lineage as was indicated by collagen type I production and alkaline phosphatase (ALP) expression. We observed no significant differences in osteogenic gene expression profiles between static and dynamic conditions like ALP, BMP2, Id1, Id2, Smad6, collagen type I, osteocalcin, osteonectin and S100A4. For the donors that showed bone formation, dynamically cultured hybrid constructs showed the same amount of bone as the statically cultured hybrid constructs. Based on these results, we conclude that a semi‐automated perfusion bioreactor system is capable of producing clinically relevant and viable amounts of human tissue‐engineered bone that exhibit bone‐forming potential after implantation in nude mice. Copyright © 2009 John Wiley & Sons, Ltd.     

Biomimetic scaffolds and dynamic compression enhance the properties of chondrocyte‐ and MSC‐based tissue‐engineered cartilage

Adult chondrocytes are surrounded by a protein‐ and glycosaminoglycan‐rich extracellular matrix and are subjected to dynamic mechanical compression during daily activities. The extracellular matrix and mechanical stimuli play an important role in chondrocyte biosynthesis and homeostasis. In this study, we aimed to develop scaffold and compressive loading conditions that mimic the native cartilage micro‐environment and enable enhanced chondrogenesis for tissue engineering applications. Towards this aim, we fabricated porous scaffolds based on silk fibroin (SF) and SF with gelatin/chondroitin sulfate/hyaluronate (SF‐GCH), seeded the scaffolds with either human bone marrow mesenchymal stromal cells (BM‐MSCs) or chondrocytes, and evaluated their performance with and without dynamic compression. Human chondrocytes derived from osteoarthritic joints and BM‐MSCs were seeded in scaffolds, precultured for 1 week, and subjected to compression with 10% dynamic strain at 1 Hz, 1 hr/day for 2 weeks. When dynamic compression was applied, chondrocytes significantly increased expression of aggrecan (ACAN) and collagen X (COL10A1) up to fivefold higher than free‐swelling controls. In addition, dynamic compression dramatically improved the chondrogenesis and chondrocyte biosynthesis cultured in both SF and SF‐GCH scaffolds evidenced by glycosaminoglycan (GAG) content, GAG/DNA ratio, and immunostaining of collagen type II and aggrecan. However, both chondrocytes and BM‐MSCs cultured in SF‐GCH scaffolds under dynamic compression showed higher GAG content and compressive modulus than those in SF scaffolds. In conclusion, the micro‐environment provided by SF‐GCH scaffolds and dynamic compression enhances chondrocyte biosynthesis and matrix accumulation, indicating their potential for cartilage tissue engineering applications.     

The activin‐βA/BMP‐2 chimera AB204 is a strong stimulator of adipogenesis

Several of the bone morphogenetic proteins (BMPs) have been reported to induce white as well as brown adipogenesis. Here, we characterized the adipogenic potential of AB204, a recombinant chimeric protein of activin‐βA and BMP‐2, in in vitro, ex vivo and in vivo settings. BMP‐2 is generally known to promote adipogenesis. When compared with BMP‐2, which previously showed varying degrees of adipogenesis, AB204 displayed superior in vitro adipogenic differentiation of mouse 3 T3‐L1 pre‐adipocytes and human adipose‐derived stem cells (hASCs). Surprisingly, implantation of hASCs, preconditioned with AB204 for as short a time as 48 h, into the subcutaneous space of athymic nude mice effectively produced fat pads, but not with BMP‐2. When BMP‐2 and AB204 were injected intraperitoneally, AB204 promoted dramatic systemic adipogenesis of C57BL/6 mice on a high‐fat diet very effectively. The results implicate the novel clinical potential of AB204, including induction of fat tissue ex vivo or in vivo for tissue re‐engineering and regenerative medicinal purposes, more than any known natural protein ligand. Copyright © 2015 John Wiley & Sons, Ltd.     

Development of a synthetic tissue engineered three‐dimensional printed bioceramic‐based bone graft with homogenously distributed osteoblasts and mineralizing bone matrix in vitro

Over the last decade there have been increasing efforts to develop three‐dimensional (3D) scaffolds for bone tissue engineering from bioactive ceramics with 3D printing emerging as a promising technology. The overall objective of the present study was to generate a tissue engineered synthetic bone graft with homogenously distributed osteoblasts and mineralizing bone matrix in vitro, thereby mimicking the advantageous properties of autogenous bone grafts and facilitating usage for reconstructing segmental discontinuity defects in vivo. To this end, 3D scaffolds were developed from a silica‐containing calcium alkali orthophosphate, using, first, a replica technique – the Schwartzwalder–Somers method – and, second, 3D printing, (i.e. rapid prototyping). The mechanical and physical scaffold properties and their potential to facilitate homogenous colonization by osteogenic cells and extracellular bone matrix formation throughout the porous scaffold architecture were examined. Osteoblastic cells were dynamically cultured for 7 days on both scaffold types with two different concentrations of 1.5 and 3 × 10⁹ cells/l. The amount of cells and bone matrix formed and osteogenic marker expression were evaluated using hard tissue histology, immunohistochemical and histomorphometric analysis. 3D‐printed scaffolds (RPS) exhibited more micropores, greater compressive strength and silica release. RPS seeded with 3 × 10⁹ cells/l displayed greatest cell and extracellular matrix formation, mineralization and osteocalcin expression. In conclusion, RPS displayed superior mechanical and biological properties and facilitated generating a tissue engineered synthetic bone graft in vitro, which mimics the advantageous properties of autogenous bone grafts, by containing homogenously distributed terminally differentiated osteoblasts and mineralizing bone matrix and therefore is suitable for subsequent in vivo implantation for regenerating segmental discontinuity bone defects. Copyright © 2016 John Wiley & Sons, Ltd.     

Development of a mechanically stable support for the osteoinductive biomaterial COLLOSS® E

The application of bone graft substitutes with osteoinductive properties is of high importance for the repair of large bone defects. COLLOSS^® E, a protein lyophilizate extracted from equine long bones, exhibits an osteoinductive potential which has been proven in several studies. In this work, a mechanically stable, but biodegradable support for COLLOSS^® E has been developed aiming at a bone graft substitute that retains shape and size when coming in contact with body fluids. Mineralization of collagen type I, isolated from horse tendon, resulted in a stable collagen hydroxyapatite nanocomposite. By means of freeze drying, this composite was used to prepare 3D scaffolds which can be filled with the cotton‐wool like COLLOSS^® E material. These scaffolds exhibit a porous microstructure and a good mechanical stability in dry and wet state. Cell culture experiments with human bone marrow stromal cells (hBMSC) revealed the cytocompatibility of the newly developed composite material. Cells were able to adhere, proliferate and differentiate into the osteoblastic lineage. The osteoinductive nature of COLLOSS^® E has been demonstrated by a significant higher activity of the osteogenic marker alkaline phosphatase (ALP) on combined scaffolds (mineralized collagen scaffolds filled with COLLOSS^® E) compared to pure scaffolds. The combination of COLLOSS^® E with scaffolds made of a collagen hydroxyapatite composite results in a synthetic bone graft substitute which can be completely remodelled into vital bone tissue opening an interesting new possibility for the therapy of bone defects. Copyright © 2008 John Wiley & Sons, Ltd.     

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

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. Copyright © 2012 John Wiley & Sons, Ltd.     

Effect of nanotubes and apatite on growth factor release from PLLA scaffolds

There is an evident clinical need for artificial bone restorative materials. In this respect, novel composites based on poly(L ‐lactic acid) (PLLA) have been described. The bone response of such polymer‐based composites is usually improved by the addition of bone morphogenetic protein‐2 (BMP‐2). However, released BMP‐2 is cleared almost immediately from the site of implantation by diffusion, whereas a prolonged retention of BMP‐2 onto the scaffold has been suggested to be more favourable. Besides the ability to improve the mechanical strength and osteoconductivity of polymeric scaffolds, both carbon nanotubes (CNTs) and microhydroxyapatite (µHA) have been described to facilitate such retention of BMP‐2 when incorporated into a composite scaffold. Therefore, in the current study, radiolabelled BMP‐2 was loaded onto plain PLLA and composite PLLA–CNT–µHA scaffolds. Subsequently, the scaffolds were implanted subcutaneously for 5 weeks in rats and BMP‐2 release was measured. Release started with an initial phase of quick release, followed by a gradual release of BMP‐2. Both scaffold types comprised the same in vivo release properties for BMP‐2. The bioactivity of the BMP‐2 remained unaltered. It can be concluded that incorporated CNTs and µHA did not affect BMP‐2 release from composite scaffold materials. Copyright © 2010 John Wiley & Sons, Ltd.     

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

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. Copyright © 2012 John Wiley & Sons, Ltd.