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Cartilage tissue engineering is the hotspot of cartilage repair. The allogenic chondrocytes appear to be a promising source of seed cells in cartilage tissue engineering. In this study, we aimed to transplant allogenic chondrocytes with chitosan hydrogel (CS)-demineralized bone matrix (DBM) hybrid scaffold (CS/DBM) to repair rabbit cartilage injury with one-step operation. After the CS/DBM scaffold was successfully fabricated, it showed that the porous CS filled the large pores of DBM, which improved the distribution of seed cells in the CS/DBM scaffold. The allogenic chondrocytes at second passage were transplanted with different scaffolds to repair rabbit cartilage injury. Twenty-four weeks after surgery, the cartilage defect in the CS/DBM group was successfully filled as shown by MRI. Moreover, the histological score of CS/DBM group was significantly higher than that of the other groups. On the aspect of biomechanical property, the regenerated cartilage in the CS/DBM group were superior to those in the other groups as determined by nanoindentation. Meanwhile, no obvious inflammatory response was observed after the transplantation of allogenic chondrocytes at 24 weeks post-surgery. Furtherly, gene expression profile for cells within the repair tissue was compared with the allogenic chondrocytes before transplantation using Agilent microarray and RT-qPCR. The results showed that some genes beneficial to cartilage regeneration, such as BMP-7, HGF, and IGF-1, were upregulated one month after transplantation. Consequently, our study demonstrated that the transplantation of allogenic chondrocytes with CS/DBM scaffold successfully repaired rabbit cartilage injury with only one-step operation, thereby providing new insights into cartilage tissue engineering.

The molecular cloning of the osteogenic proteins of the transforming growth factor-β (TGF-β) supergene family and the results of numerous pre-clinical studies in several mammalian species including non-human primates, have prematurely convinced molecular biologists, tissue engineers and skeletal reconstructionists alike to believe that single recombinant human bone morphogenetic/osteogenic proteins (hBMPs/OPs) would result in tissue induction when translated in clinical contexts. This theoretical potential has not been translated to acceptable clinical results. Clinical trials in craniofacial and orthopedic applications such as mandibular reconstruction and sinus-lift operations have indicated that supra physiological doses of a single recombinant human protein are needed to induce unacceptable tissue regeneration whilst incurring significant costs without achieving equivalence to autogenous bone grafts. The acid test for clinically relevant bone tissue engineering should now become the concept of clinically significant osteoinduction, whereby the regenerated bone is readily identifiable on radiographic examination by virtue of its opacity and trabecular architecture. The need for alternatives to the hBMPs/OPs is now felt more acutely following reported complications and performance failure associated with the clinical use of hBMP-2 and hOP-1 (BMP-7). Because of the often substandard regeneration of clinical defects implanted with hBMPs/OPs, we now need to finally deal with the provocative question: are the hBMPs/OPs the only initiators of the induction of bone formation in pre-clinical and clinical contexts? The rapid induction of bone formation by the hTGF-β₃ isoform in heteropic intramuscular sites of the Chacma baboon Papio ursinus together with TGF-β₁, TGF-β₃, BMP-2, BMP-3, OP-1, RUNX-2 and Osteocalcin up-regulation and expression, hyper cellular osteoblastic activity, osteoid synthesis, angiogenesis and capillary sprouting are the molecular and morphological foundation for the induction of bone formation in clinical contexts. The induction of bone as initiated by hTGF-β3 when implanted in the rectus abdominis muscle of P. ursinus is via the BMPs/OPs pathway with hTGF-β₃ controlling the induction of bone formation by regulating the expression of BMPs/OPs via Noggin expression, eliciting the induction of bone formation by up-regulating endogenous BMPs/OPs and it is blocked by hNoggin, providing insights into performance failure of hBMPs/OPs in clinical contexts. Physiological expression of BMPs/OPs genes upon implantation of hTGF-β₃ may escape the antagonist expression of Noggin and other inhibitors, whereas direct application of hBMPs/OPs, representing a later by-product step of the bone induction cascade as set by the TGF-β₃ master gene in primates, sets into motion Noggin' antagonist action, as shown by the limited effectiveness of hBMPs/OPs in clinical contexts. The unprecedented induction of bone formation by 250 μg hTGF-β₃ when combined with coral-derived macroporous constructs is the novel molecular and morphological frontier for the induction of bone formation in man. The induction of bone by hTGF-β₃ has been thus translated in clinical contexts to treat a large mandibular defect in a pediatric patient; 30 months after implantation of 250 μg hTGF-β₃ per gram of human demineralized bone matrix, radiographic analyses show the reconstruction of the avulsed large mandibular segment including the induction of the avulsed coronoid process.

The need for suitable bone grafts is high; however, there are limitations to all current graft sources, such as limited availability, the invasive harvest procedure, insufficient osteoinductive properties, poor biocompatibility, ethical problems, and degradation properties. The lack of osteoinductive properties is a common problem. As an allogenic bone graft, demineralized bone matrix (DBM) can overcome issues such as limited sources and comorbidities caused by invasive harvest; however, DBM is not sufficiently osteoinductive. Bone marrow has been known to magnify osteoinductive components for bone reconstruction because it contains osteogenic cells and factors. Mesenchymal stem cells (MSCs) derived from bone marrow are the gold standard for cell seeding in tissue-engineered biomaterials for bone repair, and these cells have demonstrated beneficial effects. However, the associated high cost and the complicated procedures limit the use of tissue-engineered bone constructs. To easily enrich more osteogenic cells and factors to DBM by selective cell retention technology, DBM is modified by a nanoscale self-assembling peptide (SAP) to form a composite DBM/SAP scaffold. By decreasing the pore size and increasing the charge interaction, DBM/SAP scaffolds possess a much higher enriching yield for osteogenic cells and factors compared with DBM alone scaffolds. At the same time, SAP can build a cellular microenvironment for cell adhesion, proliferation, and differentiation that promotes bone reconstruction. As a result, a suitable bone graft fabricated by DBM/SAP scaffolds and bone marrow represents a new strategy and product for bone transplantation in the clinic.

For critical size bone defects and bone non-unions, bone tissue engineering using osteoblastic differentiated adipose mesenchymal stem cells (AMSCs) is limited by the need for a biomaterial to support cell transplantation. An osteoblastic three-dimensional autologous graft made of AMSCs (3D AMSC) was developed to solve this issue. This autograft was obtained by supplementing the osteoblastic differentiation medium with demineralized bone matrix. Two surgical models were developed to assess the potential of this 3D osteogenic AMSC autograft. A four-level spinal fusion using polyetheretherketone cages was designed in six pigs to assess the early phase of ossification (8-12 weeks postimplantation). In each pig, four groups were compared: cancellous bone autograft, freeze-dried irradiated cancellous pig bone, 3D AMSC, and an empty cage. A critical size femoral defect (n = 4, bone non-union confirmed 6 months postoperatively) was used to assess the 3D AMSCs' ability to achieve bone fusion. Pigs were followed by CT scan and explanted specimens were analyzed for bone tissue remodeling by micro-CT scan, micro-radiography, and histology/histomorphometry. In the spine fusion model, bone formation with the 3D AMSC was demonstrated by a significant increase in bone content. In the critical-size femoral defect model, the 3D AMSC achieved new bone formation and fusion in a poorly vascularized fibrotic environment. This custom-made 3D osteogenic AMSC autograft is a therapeutic solution for bone non-unions and for critical-size defects.

Non-invasive bioluminescence imaging (BLI) to monitor changes in gene expression of cells implanted in live animals should facilitate the development of biomaterial scaffolds for tissue regeneration. We show that, in vitro, induction of chondrogenic differentiation in mouse bone marrow stromal cell line (CL1) and human adipose tissue derived mesenchymal stromal cells (hAMSCs), permanently transduced with a procollagen II (COL2A1) promoter driving a firefly luciferase gene reporter (PLuc) (COL2A1p·PLuc), induces PLuc expression in correlation with increases in COL2A1 and Sox9 mRNA expression and acquisition of chondrocytic phenotype. To be able to simultaneously monitor in vivo cell differentiation and proliferation, COL2A1p·PLuc labelled cells were also genetically labelled with a renilla luciferase (RLuc) gene driven by a constitutively active cytomegalovirus promoter, and then seeded in demineralized bone matrix (DBM) subcutaneously implanted in SCID mice. Non-invasive BLI monitoring of the implanted mice showed that the PLuc/RLuc ratio reports on gene expression changes indicative of cell differentiation. Large (CL1) and moderated (hAMSCs) changes in the PLuc/RLuc ratio over a 6 week period, revealed different patterns of in vivo chondrogenic differentiation for the CL1 cell line and primary MSCs, in agreement with in vitro published data and our results from histological analysis of DBM sections. This double bioluminescence labelling strategy together with BLI imaging to analyze behaviour of cells implanted in live animals should facilitate the development of progenitor cell/scaffold combinations for tissue repair.

Intervertebral disc (IVD) degeneration is the major cause of lower back pain, while the currently available treatments are symptomatic rather than curative. Tissue engineering is a powerful therapeutic strategy that can restore the normal biomechanical motion of the human spine. The ability of a biphasic elastic scaffold to structurally and elastically simulate the annulus fibrosus (AF) tissue of the IVD was explored. The outer phase of the scaffold was a ring-shaped demineralized bone matrix gelatin (BMG) extracted from cortical bone, which mimicks the type I collagen structure and ligamentous properties of outer AF. The inner phase of the scaffold was a bio-biomaterial poly(polycaprolactone triol malate) (PPCLM) orientated in concentric sheets and seeded with chondrocytes to recapitulate the inner layer of the AF, which is rich in type II collagen and proteoglycan. The mechanical properties and degradation of PPCLM could be adjusted by controlling the post-polymerization time of the pre-polymer. PPCLM also demonstrated good biocompatibility in a foreign body response in vivo assay. Incorporation of BMG into the scaffold enhanced the compressive strength compared with PPCLM alone. In addition, the tensile stress of the BMG/PPCLM scaffold was 50-fold greater than that of PPCLM alone, and close to that of normal rabbit AF. Finally, the biphasic scaffold supported the growth of rabbit chondrocytes, as confirmed by Safranin-O and type II collagen immunostaining. The excellent mechanical properties and biocompatibility of the BMG/PPCLM scaffold make it a promising candidate for AF repair.

The physiological role of cumulus cell surrounding oocytes is particularly important for normal cytoplasmic maturation of oocytes. Collagen-based demineralized bone matrix (DBM) is a valuable biomaterial for the three-dimensional (3-D) cell culture. The present study was designed to determine whether in vitro maturation (IVM) of cumulus-free oocytes in mice could be improved by using the 3-D DBM co-culture system. The results indicated that the denuded oocytes cultured in 3-D DBM co-culture system with cumulus cells showed close similarity of cortical granules (CGs) distribution pattern, had more normal maturation-promoting factor (MPF) level and zona pellucida (ZP) hardening level to the in vivo matured oocytes, and the best preimplantation development after being activated by in vitro fertilization (IVF) or parthenogenetic activation. Thus, 3-D DBM collagen scaffold could serve as a tool for fundamental in vitro studies of cells or tissues under the environment that closely assembles the in vivo conditions.

Considerable research has been focused on the development of bone morphogenetic protein-2 (BMP-2) delivery system for homologous and efficient bone regeneration. The aim of the present study was to develop a collagen-based targeting bone repair system. A collagen-binding domain (CBD) was added to the N-terminal of native BMP-2 to allow it bind to collagen specifically. We showed that the collagen-binding bone morphogenetic protein-2 (named bone morphogenetic protein2-h, BMP2-h) had maintained the full biological activity as compared to rhBMP2 lacking the CBD. In vitro functional study also demonstrated that collagen matrix could maintain higher bioactivity of BMP2-h than native BMP-2. When demineralized bone matrix (DBM) impregnated with BMP2-h was implanted subcutaneously in rats, homogeneous bone formation was observed. Moreover, in a rabbit mandible defect model, surgical implantation of collagen matrix loaded with BMP2-h exhibited remarkable osteoinductive properties and excellent homogeneous bone formation. Our studies suggested that this novel collagen-based BMP-2 targeting bone repair system induced better bone formation not only in quantity but also in quality. Similar approaches may also be used for the repair of other tissue injuries.

To develop a single-unit osteochondral tissue with demineralized bone matrix gelatin (BMG), rabbit chondrocytes were cultured on demineralized bone matrix gelatin for 6 weeks. The engineered osteochondral tissue was characterized with histology, immunolocalization, TEM, SEM, biochemical assay, and gene expression analysis. About 1.3mm viable neo-cartilage was produced on demineralized BMG. RT-PCR, immunohistochemistry, TEM, biochemical assay, and histology revealed hyaline-like cartilage with zonal layers, intense type II collagen expression, and abundant proteoglycan content formed upon BMG compared with normal cartilage. But hydroxyproline content and type I collagen gene and protein expressions were significantly lower. We consider engineering cartilage tissue with chondrocytes cultured on allogenic demineralized BMG is a good approach for osteochondral tissue engineering.

Bone substitute materials can induce bone formation in combination with mesenchymal stem cells (MSC). The aim of the current study was to examine ectopic in vivo bone formation with and without MSC on a new resorbable ceramic, called calcium deficient hydroxyapatite (CDHA). Ceramic blocks characterized by a large surface (48 m2/g) were compared with beta-tricalcium phosphate (beta-TCP), hydroxyapatite (HA) ceramics (both ca. 0.5 m2/g surface) and demineralized bone matrix (DBM). Before implantation in the back of SCID mice carriers were freshly loaded with 2x10(5) expanded human MSC or loaded with cells and kept under osteogenic conditions for two weeks in vitro. Culture conditions were kept free of xenogenic supplements. Deposits of osteoid at the margins of ceramic pores occurred independent of osteogenic pre-induction, contained human cells, and appeared in 416 MSC/CDHA composites compared to 216 MSC/beta-TCP composites. ALP activity was significantly higher in samples with MSC versus empty controls (p

The use of a composite graft of bovine trabecular demineralized bone matrix (DBM) and perichondrium has been found a reliable method for in vivo generation of cartilage. In the present study, the mechanism whereby this commercially available matrix increases cartilage formation was investigated. First, the time course of cartilage formation in vivo, in the combined implant of perichondrium and DBM in the rabbit ear was studied, with special focus on tissue reactions to DBM. DBM was colonized by macrophages from day 3 post-operatively, reaching a maximum after 2 weeks. Only a minimal number of neutrophils was found. After 3 weeks the DBM appeared to be resorbed. In the first week the DBM was invaded with chondroblasts, and chondrogenesis occurred between the first and second week of implantation. After 3 weeks, the initially formed islets of cartilage had fused. Next, the chondrogenic capacity of DBM itself was investigated by implantation of DBM without perichondrium. This never resulted in cartilage formation. Immunohistochemistry showed only a faint staining of the DBM for growth factors. This indicates a minimal chondrogenic e⁄ect of DBM alone and the requirement of perichondrium as cell provider. In order to define the conditions which cause chondrogenesis in composites of perichondrium and DBM, a series of in vitro culture experiments was performed in which the in vivo situation was mimicked step by step. The basic condition was perichondrium cultured in medium with 10% FCS. In this condition, cartilage formation was variable. Because in the in vivo situation both DBM and macrophages can release growth factors, the e⁄ect of IGF1, TGFb2 or OP1 added to the culture medium was tested. Neither the incidence nor the amount of cartilage formation was stimulated by addition of growth factors. Perichondrium wrapped around DBM in vitro gave cartilage formation in the perichondrium but the incidence and amount were not significantly stimulated compared to cultures of perichondrium without DBM. However, cartilage-like cells were found in the DBM suggesting an e⁄ect of DBM on perichondrium-derived cells. Finally, macrophages and/or blood were added to the composite DBM-perichondrium to mimic the in vivo situation as close as possible. However, no e⁄ect of this treatment was found. In conclusion, this study indicates that DBM itself has few chondrogenic qualities but functions merely as a spacer for cell ingrowth. The fast resorption of DBM by macrophages in vivo seems of importance for the cartilage forming process, but in vitro the presence of macrophages (in combination with blood) could not enhance chondrogenesis. ( 1999 Elsevier Science Ltd. All rights reserved

Autogenous bone transfer is an important part of reconstructive plastic surgery. Presently available techniques have the disadvantages of limitation of available donor site, loss of donor tissue and the possibility of donor defect or deformity. In the present study, a vascularized bone graft was created and cultured in the groin area of the New Zealand rabbit. The cylindrical ceramic chambers, 15mm in length, 6mm in outer diameter and 3mm in inner diameter, were prepared by the addition of sintered porous β-Ca2P2O7 with 5 wt% Na4P2O7-10H2O. In the first group, the chambers impregnated with autogenous bone fragments and allogenous demineralized bone matrix with volume ratio 1:1 were cultured in the rabbit's groin area with saphenous vessels passing through. In the second group, the chambers were treated by the same procedures as the first group but without saphenous vessels passing through. In the third group, the chambers were not impregnated, and were cultured in the groin area with saphenous vessels. After 2, 4, 6, 8 and 12 wk of operation, the animals were killed with an overdose of intravenous pentobarbital. The viability of the osseous tissue in the chamber was evaluated by histological examination, microangiograms and fluorochrome incorporation for the three groups. The autogenous bone chips could survive and retain their osteogenic properties while packed into the sintered porous β-Ca2P2O7 (with 5 wt% Na4P2O7 · 10H2O addition) ceramic chamber and implanted in the rabbit groin area up to 12 wk. However, even at the longest time periods, considerable amounts of dead bone were present in the chambers. In addition, we observed bone resorption in the three groups up to 12 wk, which might be attributed to lack of physiological stress. There were significant differences in new bone formation and osseous cell viability among the three groups. The prevascularized vessels and autogenous bone chips were both necessary for the formation of new bone and osteogenic property in the chamber under these heterotopic circumstances. The biodegradable ceramic used in this study was gradually absorbed and dissolved in the physiological environment. However, the degradation debris of the ceramic caused no injury to the new bone formation. These findings support the concept of creating a preformed vascularized bone graft to reconstruct segmental bone defects.

Extensive chest wall defect reconstruction remains a challenging problem for surgeons. In the past several years, little progress has been made in this area. In this study, a biodegradable polydioxanone (PDO) mesh and demineralized bone matrix (DBM) seeded with osteogenically induced bone marrow stromal cells (BMSCs) were used to reconstruct a 6 cm x 5.5 cm chest wall defect. Four experimental groups were evaluated (n=6 per group): polydioxanone (PDO) mesh/DBMs/BMSCs group, polydioxanone (PDO) mesh/DBMs group, polydioxanone (PDO) mesh group, and a blank group (no materials) in a canine model. All the animals survived except those in the blank group. In all groups receiving biomaterial implants, the polydioxanone (PDO) mesh completely degraded at 24 weeks and was replaced by fibrous tissue with thickness close to that of the normal intercostal tissue (P>0.05). In the polydioxanone (PDO) mesh/DBMs/BMSCs group, new bone formation and bone-union were observed by radiographic and histological examination. More importantly, the reconstructed rib could maintain its original radian and achieve satisfactory biomechanics close to normal ribs in terms of bending stress (P>0.05). However, in the other two groups, fibrous tissue was observed in the defect and junctions, and the reconstructed ribs were easily distorted under an outer force. Based on these results, a surgical approach utilizing biodegradable polydioxanone (PDO) mesh in combination with DBMs and BMSCs could repair the chest wall defect not only in function but also in structure.

Demineralized bone matrix (DBM) is a collagen-based scaffold, but its low mechanical strength and limited BMP-2 binding ability restrict its application in bone repair. It is known that heparin could be immobilized onto scaffolds to enhance their binding of growth factors with the heparin-binding domain. Here, we crosslinked heparin to DBM to increase its BMP-2 binding ability. To our surprise, the mechanical strength of DBM was also dramatically increased. The compression modulus of heparin crosslinked DBM (HC-DBM) have improved (seven-fold increased) under wet condition, which would allow the scaffolds to keep specific shapes in vivo. As expected, HC-DBM showed specific binding ability to BMP-2. Additional studies showed the bound BMP-2 exerted its function to induce cell differentiation on the scaffold. Subcutaneous implantation of HC-DBM carrying BMP-2 showed higher alkaline phosphatase (ALP) activity (2 weeks), more calcium deposition (4 and 8 weeks) and more bone formation than that of control groups. It is concluded that HC-DBM has increased mechanical intensity as well as specific BMP-2 binding ability; HC-DBM/BMP-2 enhances the osteogenesis and therefore could be an effective medical device for bone repair.

The aim of this study was to compare three resorbable biomaterials regarding seeding efficacy with human bone marrow stromal cells (BMSCs), cell penetration into the matrix, cell proliferation and osteogenic differentiation. Calcium-deficient hydroxyapatite (CDHA), beta-tricalcium phosphate (beta-TCP), and demineralized bone matrix (DBM) were seeded with human BMSCs and kept in human serum and osteogenic supplements for 3 weeks. Morphologic and biochemical evaluations were performed on day 1, 7, 14 and 21. The allograft DBM and CDHA exhibited both an excellent seeding efficacy while the performance of beta-TCP was lower when compared. The total protein content and the values for specific alkaline phosphatase (ALP) increased on all matrices and no significant difference was found for these two markers. BMSCs in monolayer had a significant increase of protein, but not of ALP. Osteocalcin (OC) values increased significantly higher for BMSC in cultures on DBM when compared to CDHA and beta-TCP. The OC levels decreased significantly in the BMSC monolayer culture. BMSCs were found inconsistently within the synthetic materials, whereas in DBM they were found more homogeneously distributed throughout the matrix. All three matrices promoted BMSC proliferation and differentiation to osteogenic cells. DBM allografts seem to be more favorable with respect to cell ingrowth tested by histology, and osteogenic differentiation ascertained by an increase of OC. CDHA with its high specific surface area showed more favorable properties than beta-TCP regarding reproducibility of the seeding efficacy.

The effects of different biomaterials, used in orthopaedic surgery for bone substitution and/or prosthesis coating to improve fixation and durability of prosthetic implants, have been studied in vitro on cell growth and bone matrix synthesis by human osteoblasts. The materials were a bovine collagen matrix (Osteovit, B. Braun Melsungen AG, Melsungen, Germany) and two hydroxyapatite (Ceros 80, Robert Mathys Co, Instrumentenfabrik Bettlach, Germany and Ostilit, Howmedica International, Staines House, UK). Cell proliferation and bone matrix synthesis were assessed by incorporation of [3H]thymidine and [3H]proline, respectively. Cell viability in the presence of the materials was also morphologically controlled using phase-contrast microscopy. Exposure to Osteovit caused increased proliferation of human osteoblasts, whereas both Ostilit and Ceros 80 induced a decreased cell growth. Osteoblast bone matrix synthesis was increased by all the materials tested.

The replacement of bone defects is very important in clinical practice. This study compares biological properties of poly(2-hydroxyethyl methacrylate)-collagen composite with those exhibited by pure p(HEMA), an insoluble fraction of calf skin collagen (ISC-40) and demineralized bone matrix after implantation into pig or dog femurs. The levels of biodegradation or destruction of implants and healing of bone defects were studied using X-ray photography, histology and enzyme histochemistry. The results indicated a significant effect of collagen on biological destruction of the p(HEMA)-composite implants; even a minute amount of collagen influences this process dramatically. A stimulatory action of collagen on new bone formation may be of importance in bone defect healing.

Teflon tubes with either Gore-Tex (PTFE) or Dacron felt sleeves were implanted percutaneously in rats, in conjunction with demineralized bone matrix. This resulted in different inflammatory reactions, as well as in chondrogenesis and osteogenesis in the subcutaneous tissues. Although induction of osteogenesis by the demineralized bone matrix in the vicinity of the foreign material was inhibited, circumferential bone formation was highly reproducible. The prospect of utilizing demineralized bone matrix in order to enhance acceptance of percutaneous tubes is discussed.