Delivery of recombinant bone morphogenetic proteins for bone regeneration and repair. Part A: Current challenges in BMP delivery

Recombinant human bone morphogenetic proteins (rhBMPs) have been extensively investigated for developing therapeutic strategies aimed at the restoration and treatment of orthopaedic as well as craniofacial conditions. In this first part of the review, we discuss the rationale for the necessary use of carrier systems to deliver rhBMP-2 and rhBMP-7 to sites of bone tissue regeneration and repair. General requirements for growth factor delivery systems emphasizing the distinction between localized and release-controlled delivery strategies are presented highlighting the current limitations in the development of an effective rhBMP delivery system applicable in clinical bone tissue engineering.     

Delivery of bone morphogenetic proteins for orthopedic tissue regeneration

Carriers for bone morphogenetic proteins (BMPs) are used to increase retention of these factors at orthopedic treatment sites for a sufficient period of time to allow regenerative tissue forming cells to migrate to the area of injury and to proliferate and differentiate. Carriers can also serve as a matrix for cell infiltration while maintaining the volume in which repair tissue can form. Carriers have to be biocompatible and are often required to be bioresorbable. Carriers also have to be easily, and cost-effectively, manufactured for large-scale production, conveniently sterilized and have appropriate storage requirements and stability. All of these processes have to be approvable by regulatory agencies. The four major categories of BMP carrier materials include natural polymers, inorganic materials, synthetic polymers, composites of these materials. Autograft or allograft carriers have also used. Carrier configurations range from simple depot delivery systems to more complex systems mimicking the extracellular matrix structure and function. Bone regenerative carriers include depot delivery systems for fracture repair, three-dimensional polymer or ceramic composites for segmental repairs and spine fusion and metal or metal/ceramic composites for augmenting implant integration. Tendon/ligament regenerative carriers range from depot delivery systems to three-dimensional carriers that are either randomly oriented or linearly oriented to improve regenerative tissue alignment. Cartilage regenerative systems generally require three-dimensional matrices and often incorporate cells in addition to factors to augment the repair. Alternative BMP delivery systems include viral vectors, genetically altered cells, conjugated factors and small molecules.     

The application of bone morphogenetic proteins to dental tissue engineering

Progress in understanding the role of bone morphogenetic proteins (BMPs) in craniofacial and tooth development, the demonstration of stem cells in dental pulp and accumulating knowledge on biomaterial scaffolds have set the stage for tissue engineering and regenerative therapy of the craniofacial complex. Furthermore, the recent approval by the US Food and Drug Administration (FDA; Rockville, MD, USA) of recombinant human BMPs for accelerating bone fusion in slow-healing fractures indicates that this protein family may prove useful in designing regenerative treatments in dental applications. In the near term, these advances are likely to be applied to endodontics and periodontal surgery; ultimately, they may facilitate approaches to regenerating whole teeth for use in tooth replacement.     

Recent progress in bone induction by osteogenin and bone morphogenetic proteins: challenges for biomechanical and tissue engineering

Implantation of demineralized bone matrix results in local bone induction. Bone induction is a sequential biological chain reaction that consists of chemotaxis and proliferation of mesenchymal cells and differentiation of bone. Osteogenin, a bone morphogenetic protein has been purified and the amino acid sequence determined. Recently a family of bone morphogenetic proteins have been cloned and expressed by recombinant DNA technology. The availability of growth and morphogenetic factors will permit the rational design of new bone. The challenge for the biomechanical engineer is to attain mechanically optimal and functionally adaptive new bone for various skeletal prostheses. We are on the threshold for fabrication of new bone based on sound architectural design principles of tissue engineering based on cellular and molecular biology of growth and differentiation factors.     

Prediction of heparin binding sites in bone morphogenetic proteins (BMPs)

Heparin is a glycosaminoglycan known to bind bone morphogenetic proteins (BMPs) and the growth and differentiation factors (GDFs) and has strong and variable effects on BMP osteogenic activity. In this paper we report our predictions of the likely heparin binding sites for BMP-2 and 14. The N-terminal sequences upstream of TGF-β-type cysteine-knot domains in BMP-2, 7 and 14 contain the basic residues arginine and lysine, which are key components of the heparin/HS-binding sites, with these residues being highly non-conserved. Importantly, evolutionary conserved surfaces on the beta sheets are required for interactions with receptors and antagonists. Furthermore, BMP-2 has electropositive surfaces on two sides compared to BMP-7 and BMP-14. Molecular docking simulations suggest the presence of high and low affinity binding sites in dimeric BMP-2. Histidines were found to play a role in the interactions of BMP-2 with heparin; however, a pK(a) analysis suggests that histidines are likely not protonated. This is indicative that interactions of BMP-2 with heparin do not require acidic pH. Taken together, non-conserved amino acid residues in the N-terminus and residues protruding from the beta sheet (not overlapping with the receptor binding sites and the dimeric interface) and not C-terminal are found to be important for heparin-BMP interactions.     

Bone morphogenetic proteins in craniofacial and periodontal tissue engineering: experimental studies in the non-human primate Papio ursinus

The bone morphogenetic and osteogenic proteins (BMPs/OPs), pleiotropic members of the transforming growth factor-beta (TGF-beta) supergene family act as soluble signals for the de novo initiation of bone formation, sculpting the multicellular mineralized structures of the bone-bone marrow organ. The strikingly pleiotropic effects of BMPs/OPs spring from amino acid sequence variations in the carboxy-terminal domain and in the transduction of distinct signalling pathways by individual Smad proteins after transmembrane serine/threonine kinase complexes of type I and II receptors. BMPs/OPs are the common molecular initiators deployed for embryonic development and the induction of bone formation and regeneration in postnatal osteogenesis. Naturally derived BMPs/OPs extracted and purified from baboon and bovine bone matrices induce complete regeneration of non-healing calvarial defects in the non-human primate Papio ursinus as well as the induction of cementogenesis and the morphogenesis of a periodontal ligament system with a faithful insertion of Sharpey's fibers into the newly formed cementum. gamma-Irradiated recombinant human osteogenic protein-1 (hOP-1) delivered by xenogeneic bovine collagenous bone matrices completely regenerated and maintained the architecture of the induced bone after treatment of calvarial defects with single applications of doses of 0.1, 0.5 and 2.5mg hOP-1 per gram of carrier matrix. The long-term implantation of hOP-1 delivered by gamma-irradiated bovine bone matrices induced the regeneration of the three essential components of the periodontium, i.e. cementum, periodontal ligament and alveolar bone. The osteogenic proteins of the TGF-beta superfamily are sculpting tissue constructs that engineer skeletal tissue regeneration in molecular terms. The pleiotropy of the signalling molecules of the TGF-beta superfamily is highlighted by the redundancy of molecular signals initiating bone formation, including the TGF-beta isoforms per se, powerful inducers of endochondral bone formation but in the primate only. The induction of bone develops a mosaic structure in which members of the TGF-beta superfamily singly, synergistically and synchronously initiate and maintain tissue induction and morphogenesis.     

Biodegradable bone regeneration synthetic scaffolds: in tissue engineering

A growing array of synthetic bone regeneration scaffolds has been used or investigated over the last century. These scaffolds aim to provide a three dimensional substrate for bone cells to populate on and function appropriately. To serve this function, these scaffolds should be biocompatible and biodegradable at a rate commensurate with bone remodelling. Their mechanical properties should also be similar to those of the bone regeneration site. In this review, the main families of synthetic bone scaffolds were taxonomised and expounded. The main focus of this paper will be on the basic sciences principles and properties of clinical available as well as experimental synthetic bone scaffolds. Special emphasis was put on scaffolds developed over the last ten years.     

Protein- and gene-based tissue engineering in bone repair

A tissue engineering approach to bone regeneration includes the use of a scaffold, cells and bioactive factors alone or in various combinations. Several investigators have demonstrated enhanced bone formation when the tissue-engineered construct possesses traits inherent to autogenic bone grafts, namely osteoconductivity, osteoinductivity and osteogenicity. Use of the biodegradable polymer poly(lactide-co-glycolide) in combination with bone morphogenetic protein or primary cells genetically modified to release osteogenic protein have demonstrated the ability to induce osteogenic differentiation of, and subsequent mineralization by, muscle-derived cells and mesenchymal stem cells in both in vitro and in vivo applications.     

Protein-based tissue engineering in bone and cartilage repair

Bioactive proteins signal host or transplanted cells to form the desired tissue type. Matrix systems are utilized to locally deliver the proteins and to maintain effective protein concentrations. For some indications, a matrix is required to define the physical form of the regenerated tissue. Substantial progress has been made in bone tissue engineering in recent years, based on the results of controlled clinical studies using bone morphogenetic proteins. Ongoing research in this area centers on the design of additional delivery matrices to expand the clinical indications, using synthetic delivery systems that mimic biological qualities of the natural materials currently in use. Although a similar rationale exists for the regeneration of articular cartilage with bioactive factors, advancement in this area has not been as substantial.     

Perinatal stem cells: A promising cell resource for tissue engineering of craniofacial bone

In facing the mounting clinical challenge and suboptimal techniques of craniofacial bone defects resulting from various conditions, such as congenital malformations, osteomyelitis, trauma and tumor resection, the ongoing research of regenerative medicine using stem cells and concurrent advancement in biotechnology have shifted the focus from surgical reconstruction to a novel stem cell-based tissue engineering strategy for customized and functional craniofacial bone regeneration. Given the unique ontogenetical and cell biological properties of perinatal stem cells, emerging evidence has suggested these extraembryonic tissue-derived stem cells to be a promising cell source for extensive use in regenerative medicine and tissue engineering. In this review, we summarize the current achievements and obstacles in stem cell-based craniofacial bone regeneration and subsequently we address the characteristics of various types of perinatal stem cells and their novel application in tissue engineering of craniofacial bone. We propose the promising feasibility and scope of perinatal stem cell-based craniofacial bone tissue engineering for future clinical application.     

Use of bone morphogenetic proteins in mesenchymal stem cell stimulation of cartilage and bone repair

The extracellular matrix-associated bone morphogenetic proteins(BMPs) govern a plethora of biological processes. The BMPs are members of the transforming growth factor-β protein superfamily, and they actively participate to kidney development, digit and limb formation, angiogenesis, tissue fibrosis and tumor development. Since their discovery, they have attracted attention for their fascinating perspectives in the regenerative medicine and tissue engineering fields. BMPs have been employed in many preclinical and clinical studies exploring their chondrogenic or osteoinductive potential in several animal model defects and in human diseases. During years of research in particular two BMPs, BMP2 and BMP7 have gained the podium for their use in the treatment of various cartilage and bone defects. In particular they have been recently approved for employment in non-union fractures as adjunct therapies. On the other hand, thanks to their potentialities in biomedical applications, there is a growing interest in studying the biology of mesenchymal stem cell(MSC), the rules underneath their differentiation abilities, and to test their true abilities in tissue engineering. In fact, the specific differentiation of MSCs into targeted celltype lineages for transplantation is a primary goal of the regenerative medicine. This review provides an overview on the current knowledge of BMP roles and signaling in MSC biology and differentiation capacities. In particular the article focuses on the potential clinical use of BMPs and MSCs concomitantly, in cartilage and bone tissue repair.     

Tissue engineering: TGF-beta superfamily members and delivery systems in bone regeneration

The induction of bone formation requires three parameters that interact in a highly regulated process: soluble osteoinductive signals, capable responding cells, and a supporting matrix substratum or insoluble signal. The use of recombinant and naturally derived bone morphogenetic proteins and transforming growth factor beta(s) (TGF-beta(s)) has increased our understanding of the functions of these morphogens during the induction of endochondral bone formation. In addition, growing understanding of the cellular interactions of living tissues with synthetic biomaterials has led to the in vivo induction of bone formation using porous biomimetic matrices as an alternative to the use of autografts for bone regeneration. This review outlines the basis of bone tissue engineering by members of the TGF-beta superfamily, focusing on their delivery systems and the intrinsic induction of bone formation by specific biomimetic matrices with a defined geometry.     

Regulation of cartilage and bone differentiation by bone morphogenetic proteins.

Quantum advances have recently been made in the understanding of the regulation of cartilage and bone differentiation through the identification, purification, genetic cloning and expression of recombinant bone morphogenetic proteins. Bone morphogenetic proteins are a family of pleiotropic differentiation factors with actions on chemotaxis, mitosis, initiation and promotion of chondrogenic and osteogenic phenotypes. They bind extracellular matrix components, heparin and type IV collagen and initiate bone repair. The cascade of cartilage and bone differentiation consists of several continuous phases: initiation, promotion, maintenance and termination.     

Combinatorial gene therapy for bone regeneration: cooperative interactions between adenovirus vectors expressing bone morphogenetic proteins 2, 4, and 7

Bone morphogenetic proteins (BMPs) have demonstrated effectiveness as bone regeneration agents whether delivered as recombinant proteins or via gene therapy. Current gene therapy approaches use vectors expressing single BMPs. In contrast, multiple BMPs are coordinately expressed during bone development and fracture healing. Furthermore, BMPs likely exist in vivo as heterodimeric molecules having enhanced biological activity. In the present study, we test the hypothesis that gene therapy-based bone regeneration can be enhanced by expressing combinations of BMPs. For in vitro studies, mesenchymal cell lines were transduced with individual adenoviruses containing BMP2, 4, or 7 cDNA under control of a CMV promoter (AdBMP2, 4, 7) or virus combinations. Significantly, combined transduction with AdBMP2 plus AdBMP7 or AdBMP4 plus AdBMP7 resulted in a synergistic stimulation of osteoblast differentiation. This synergy is best explained by formation of BMP2/7 and 4/7 heterodimers. To test in vivo biological activity, fibroblasts were transduced with specific virus combinations and implanted into C57BL6 mice. Consistent with in vitro results, strong synergy was observed using combined AdBMP2/BMP7 treatment, which induced twofold to threefold more bone than would be predicted based on the activity of individual AdBMPs. These studies show that dramatic enhancement of osteogenesis can be achieved using gene therapy to express specific combinations of interacting regenerative molecules.