Heparan-like molecules induce the repair of skull defects

Heparin-binding growth factors (HBGFs) are known to stimulate bone repair when applied to bone lesions. Nevertheless, successful treatments are obtained with high protein doses since HBGFs are rapidly degraded in situ by multiple proteolytic activities associated with the inflammatory period of tissue healing. Like heparin or heparan sulfates, heparan-like molecules, named carboxymethyl-benzylamide-sulfonated dextrans (CMDBS), are known to potentiate fibroblast growth factor activities by stabilizing them against pH, thermal or proteolytic denaturations, and by enhancing their binding with cell surface receptors. We have postulated that CMDBS stimulate in vivo bone healing by interacting with endogenous HBGFs, spontaneously released in the wounded site. The effect of CMDBS on bone repair was studied in a skull defect model in rats by computer-assisted radiomorphometry and histomorphometry. Single application of CMDBS in a collagen vehicle to skull defects induced a dose-dependent increase in bone defect closure and new bone formation after 35 days. Complete bony bridging occurred in defects treated with 3 μg CMDBS, whereas bone formation was not observed in vehicle-treated defects which contained only dense fibrous connective tissue between the defect margins. These results indicate that heparan-like molecules, such as CMDBS, are able to induce bone regeneration of skull defects. This action is possibly mediated by potentiation of endogenous growth factor activities and/or by neutralization of proteolytic activities.     

Regulation and guidance of cell behavior for tissue regeneration via the siRNA mechanism

RNA intereference and short-interfering RNA (siRNA) have been proven to be effective at decreasing the expression of target genes and provide a valuable tool for promoting and directing the growth of functional tissues for repair and reconstructive tissue engineering applications. siRNA is a gene-silencing mechanism that involves double-stranded RNA-mediated sequence-specific mRNA degradation and is a powerful mechanism for controlling cell behavior. The use of siRNA to reduce the expression of a target gene can induce the expression of one or more tissue-inductive factors, direct the differentiation of stem or progenitor cells, or remove a factor that inhibits regeneration, which can be useful in fundamental studies of tissue formation or in applications to promote in vivo regeneration. The potential of siRNA is illustrated through specific examples within the fields of angiogenesis, bone and nerve regeneration, and wound healing. In addition, challenges to deliver siRNA effectively for tissue engineering applications are addressed. siRNA represents a powerful tool to investigate and/or promote tissue formation, and numerous opportunities exist for identifying targets that promote regeneration of tissue and developing effective delivery systems.     

Reviewing the structural features of autologous platelet-leukocyte gel and suggestions for use in surgery

The therapeutic use of autologously prepared platelet-leukocyte gel (PLG) is a relatively new technology which might stimulate and accelerate soft-tissue and bone healing. The effectiveness of this procedure lies in the exogenous delivery of a wide range of platelet growth factors, intentionally released from autologously prepared PLG. The rationale to employ this technique is to mimic physiological wound healing and reparative tissue processes. Despite an increase in clinical PLG applications, the structures and kinetics of this biological material have not been completely examined. Electron microscopic imaging was performed to evaluate platelet-leukocyte gel structures. Furthermore, directions for PLG application are presented, based on results from published articles in various surgical disciplines. In conclusion, PLG can be useful in a wide range of clinical applications to enhance healing following surgical procedures, since exogenous applied PLG releases instantly platelet growth factors, in the presence of leukocytic cells.     

Growth factors in bone

Bone contains several growth factors, including bone morphogenetic proteins (BMPs), transforming growth factor beta (TGF-beta), insulin-like growth factors I and II (IGF-I and IGF-II), platelet derived growth factor (PDGF) and basic and acidic fibroblast growth factor (bFGF and aFGF). Spatial and temporal variations in the expression and secretion of the various growth factors have been demonstrated in osteoblastic cultures and in various experimental and clinical in vivo models, including fracture healing in humans. Local application of various growth factors influences proliferation, differentiation and protein synthesis in osteoblastic cultures and bone formation in different animal models, including experimental fractures and skeletal defects. The BMPs are the only growth factors known to provoke bone formation heterotopically by making undifferentiated mesenchymal cells differentiate into osteoblasts (osteoinduction). BMPs and other growth factors, soon to become commercially available for clinical use, need a delivery system for their sustained release, as the factors are otherwise rapidly absorbed. Some existing systems inhibit bone formation by inducing chronic inflammation or physically by unresorbed carrier obstructing bone formation. New delivery systems are being investigated.     

Gene expression of insulin-like growth factors I and II in rat membranous osteotomy healing.

Poorly healing mandibular osteotomies can be a difficult problem in reconstructive surgery. Many therapies have been attempted to augment the healing of mandibular fractures, defects, or osteotomies, but these methods have substantial drawbacks or have been ineffective. The difficulty in treating poorly healing bony defects has led to the exploration of gene therapy as a possible approach to supplement or accelerate mandibular fracture healing. To understand at what point the introduction of a suitable gene candidate might be of benefit in mandibular healing, it is imperative to examine the temporal expression of bone growth factors in a model of membranous bone healing. Insulinlike growth factors (IGFs) I and II are two such bone growth factor candidates because of their known potent in vitro as well as in vivo effects on bone formation. In this study the authors demonstrate the temporal pattern of IGF I and IGF II gene expression during mandibular osteotomy healing using a rat model. Their data reveal that IGF I and IGF II were elevated 7 days after a mandibular osteotomy that was held in external fixation. The upregulation of IGF I and IGF II during mandibular bone healing underscores the importance of these growth factors in bone repair. Gene therapy utilizing recombinant viral constructs containing IGFs I and II may be of benefit during mandibular bone healing in an effort to augment clinical scenarios of poor or retarded bony repair.     

Carrier systems and application of growth factors in orthopaedics

SUMMARY: With optimal surgical treatment within an appropriate time frame, bony tissue has the potential to regenerate defects without the formation of scar tissue. However, even under optimal mechanical circumstances and appropriate operative treatment, healing can fail and delayed or non-union occur. In Europe delayed bone healing leads to socio-economic costs of up to 14.7 billion euros per year. In addition to the optimal clinical treatment, the success of bone regeneration depends on the following main aspects: (1) adequate mechanical stabilization and biological competence of the organism, (2) osteogenic cells, (3) osteoconductive structures or scaffolds, and (4) growth factors (Diamond Concept)(1). Further, (5) a sufficient vascularisation is essential for the nutritive supply. Within the last years two growth factors, BMP-2 and BMP-7, were approved for clinical use in orthopaedic and trauma surgery for different indications.(2,3) The establishment of carrier systems and application techniques for growths factors is the focus of current research. The combination of a well established stabilization system and local drug delivery system for bioactive factors could be a therapeutical strategy to optimize bone healing and reduce the complication rate, in the future.     

The use of autologous platelet–leukocyte gels to enhance the healing process in surgery,a review

Background The therapeutic use of autologously prepared, platelet–leukocyte-enriched gel (PLG) is a relatively new technology for the stimulation and acceleration of soft tissue and bone healing. The effectiveness of this procedure lies in the delivery of a wide range of platelet growth factors mimicking the physiologic wound healing and reparative tissue processes. Despite an increase in PLG applications, the structures and kinetics of this autogenously derived biologic material have not been observed. Methods A review of the most recent literature was performed to evaluate the use of PLG in various surgical disciplines. Results The review showed that the application of PLG has been extended to various surgical disciplines including orthopedics, cardiac surgery, plastic and maxillofacial surgery, and recently also endoscopic surgery. Conclusion This review demonstrates the usefulness of PLG in a wide range of clinical applications for improvement of healing after surgical procedures.     

Platelet-Rich Plasma for the Replenishment of Bone

This review examines the use of platelet-rich plasma (PRP) in the treatment of bone injuries and to stimulate bone formation. Studies examining both in vivo bone healing and in vitro actions of PRP on osteoblasts are reviewed. Overall, the available literature suggests that PRP does not appreciably impact bone healing or induce bone formation. However, there is some evidence to suggest that PRP might augment recruitment of osteoblast progenitors to injection sites or in sites expected to experience delayed healing. In this capacity PRP might be utilized to initiate repair of an otherwise poorly healing skeletal lesion. The demonstration that PRP is a viable therapy is hindered by a lack of standardized criteria for what constitutes PRP, and more studies are needed to compare the efficacy of PRP to that of transforming growth factor-β or platelet-derived growth factor used as sole agents.     

Engineered decellularized matrices to instruct bone regeneration processes

Despite the significant progress in the field of bone tissue engineering, cell-based products have not yet reached the stage of clinical adoption. This is due to the uncertain advantages from the standard-of-care, combined with challenging cost-and regulatory-related issues. Novel therapeutic approaches could be based on exploitation of the intrinsic regenerative capacity of bone tissue, provided the development of a deeper understanding of its healing mechanisms. While it is well-established that endogenous progenitors can be activated toward bone formation by overdoses of single morphogens, the challenge to stimulate the healing processes by coordinated and controlled stimulation of specific cell populations remains open. Here, we review the recent approaches to generate osteoinductive materials based on the use of decellularized extracellular matrices (ECM) as reservoirs of multiple factors presented at physiological doses and through the appropriate ligands. We then propose the generation of customized engineered and decellularized ECM (i) as a tool to better understand the processes of bone regeneration and (ii) as safe and effective “off-the-shelf” bone grafts for clinical use.This article is part of a Special Issue entitled Stem Cells and Bone.     

Bone challenges for the hand surgeon: from basic bone biology to future clinical applications

Bone is a complex tissue composed of a calcified extracellular matrix with specialized cells that produce, maintain, and resorb the bone. Bone also has a rich vascular and neural supply. Bone has a great capability of regeneration, healing, and remodelling that is influenced by external factors, such as stress forces, and internal regulators that include hormones, vitamins, and growth factors. These factors dictate bone biology, and variations result in pathophysiologic conditions that have clinical implications in hand surgery. Solutions to the challenges in hand surgery rely on a thorough understanding of the biology of bone.     

What should be the characteristics of the ideal bone graft substitute? Combining scaffolds with growth factors and/or stem cells

Reconstruction of large bone defects or non-unions resulting from biochemical disorders, tumour resections or complicated fractures is still a challenge for orthopaedic and trauma surgery. On the one hand, autografts harbour most features of ideal bone graft substitutes but on the other hand, they have a lot insurmountable disadvantages. An ideal bone graft substitute should be biomechanically stable, able to degrade within an appropriate time frame, exhibit osteoconductive, osteogenic and osteoinductive properties and provide a favourable environment for invading blood vessels and bone forming cells. Whilst osteoconductivity of biomaterials for bone tissue engineering strategies can be directed by their composition, surface character and internal structure, osteoinductive and osteogenic features can be provided by growth factors originally participating in fracture healing and/or multipotent mesenchymal stromal/stem cells (MSC) capable of rebuilding bone and marrow structures. In this review, aspects of the clinical application of the most commonly used growth factors for bone repair, the bone morphogenetic proteins (BMPs), and the potential use of human MSC for clinical application will be discussed.     

骨折愈合、延迟愈合和骨不连

骨折愈合分为四个阶段：血肿炎症期、肉芽形成期、骨化期和重塑期。四阶段的病理变化连续而重叠，诸多炎性介质和生长因子起了非常重要的作用。最终骨折根据稳定性程度达到直接愈合或骨痂愈合。骨折是否发生延迟愈合和骨不连可以从治疗时间、临床表现、放射学表现和生物力学几方面进行评价，重点对骨不连的两种基本形式进行了解释，并对造成延迟愈合和骨不连的主要原因进行了分析，并从生物性、全身性、机械性和生物物理治疗四方面概括了促进骨折愈合的多种手段。     

Bringing new life to damaged bone: The importance of angiogenesis in bone repair and regeneration

Bone has the unique capacity to heal without the formation of a fibrous scar, likely because several of the cellular and molecular processes governing bone healing recapitulate the events during skeletal development. A critical component in bone healing is the timely appearance of blood vessels in the fracture callus. Angiogenesis, the formation of new blood vessels from pre-existing ones, is stimulated after fracture by the local production of numerous angiogenic growth factors. The fracture vasculature not only supplies oxygen and nutrients, but also stem cells able to differentiate into osteoblasts and in a later phase also the ions necessary for mineralization.This review provides a concise report of the regulation of angiogenesis by bone cells, its importance during bone healing and its possible therapeutic applications in bone tissue engineering. This article is part of a Special Issue entitled “Stem Cells and Bone”.     

Osteogenic protein-1 in knee arthritis and arthroplasty

The use of graft materials to restore bone stock and promote healing and implant stabilization is a crucial part of total knee arthroplasty, especially in revision surgery. Recent research has centered on the use of osteoinductive materials to promote bone formation. Osteogenic proteins are members of a superfamily of proteins called transforming growth factor-beta that, either alone or in combination with other regulatory molecules, induce new bone formation. The cloning and genetic expression of recombinant human osteogenic proteins has led to production of quantities sufficient for their clinical use. Recombinant human osteogenic protein-1 has been combined with bone-derived Type I collagen for delivery to an implant site. Preclinical studies have shown that the osteoinductive capacity of autograft and allograft bone and bone graft substitute materials can be notably improved with the addition of osteogenic protein-1. The use of this protein consistently improved the amount and rate of new bone formation compared with graft alone, resulting in earlier graft incorporation and consolidation. In addition, because osteogenic proteins are chondrogenic, they also may have a role in the treatment of cartilage injury and degeneration. Osteogenic protein-1 has been shown to induce hyalinelike cartilage repair of full thickness osteochondral defects in animal models with no degradation of the tissue with time. Although no detailed clinical studies in knee surgery have been reported with the use of osteogenic protein-1, in anecdotal cases its use alone and with bone graft materials indicate results consistent with those obtained in preclinical studies.     

Healing modulation induced by freeze-dried platelet-rich plasma and micronized allogenic dermis in a diabetic wound model

The incidence and prevalence of chronic and diabetic wounds are increasing and clinical treatments to tackle these epidemics are still insufficient. In this study, we tested the ability of freeze‐dried platelet‐rich plasma (PRP) and an allogenic micronized acellular dermal matrix alone and in combination to modulate diabetic wound healing. Therapeutic materials were applied to 1.0 cm² excisional wounds on genetically diabetic (db/db) mice. Wound‐healing kinetics and new tissue formation were studied at 9 and 21 days posttreatment. Quantitative immunohistochemistry was used to study vascularity and cellular proliferation (days 9 and 21), and collagen deposition was evaluated 21 days postwounding. In vitro, micronized allogenic dermis, when combined with PRP, absorbed nearly 50% of original platelet‐derived growth factor, transforming growth factor‐β, vascular endothelial growth factor, and epidermal growth factor from platelets and stimulated fibroblast proliferation. In vivo, micronized dermis increased the formation of vascularized wound tissue by day 9. Freeze‐dried PRP alone or in combination with micronized dermis increased wound tissue revascularization and proliferation compared with spontaneous healing. The increase in cell proliferation persisted until day 21 only when freeze‐dried PRP was used in combination with micronized dermis. These results indicate that micronized allogenic dermis may be used to provide a dermal matrix to stimulate tissue formation and the combination with PRP may confer additional beneficial growth factors to chronic or diabetic wounds.     

The role of electrical stimulation in bone repair

Electric and electromagnetic fields regulate extra-cellular matrix synthesis and stimulate repair of fractures and nonunions. Studies of electric and electromagnetic fields suggest they (1) regulate proteoglycan and collagen synthesis and increase bone formation in models of endochondral ossification, (2) accelerate bone formation and repair, (3) increase union rates in fractures previously refractory to healing, and (4) produce results equivalent to bone grafts. Electric and electromagnetic fields regulate the expression of genes in connective tissue cells for extra-cellular matrix proteins, which results in an increase in cartilage and bone. They also increase gene expression for and synthesis of growth factors, which may be an intermediary mechanism of activity and may amplify field effects through autocrine and paracrine signaling.     

The combination of platelet-derived growth factor-BB and insulin-like growth factor-I stimulates bone repair in adult Yucatan miniature pigs

The combination of insulin-like growth factor-I and platelet-derived growth factor-BB has previously been shown to stimulate healing of soft tissue wounds and the formation of bone and ligament around teeth. The purpose of the present study was to evaluate the effects of platelet-derived growth factor-BB and insulin-like growth factor-I individually and in combination on the healing of osseous wounds. Four standardized cortical wounds were created in each tibia of 11 adult Yucatan miniature pigs. The wounds in one tibia per animal were treated with either purified recombinant human insulin-like growth factor-I, platelet-derived growth factor-BB, or both in a methylcellulose gel. The wounds in each contralateral tibia received placebo gel alone. Coded serial sections of each wound were evaluated by computer-aided histomorphometry 21 days after surgery. The area and perimeter of the newly formed mineralized callus, the thickness of the total callus, and the percentage of mineralized tissue within the callus were significantly increased compared with the values of matched controls only in wounds treated with a combination of insulin-like growth factor-I and platelet-derived growth factor-BB. No significant differences in the measured parameters of callus formation were found in wounds treated with either insulin-like growth factor-I or platelet-derived growth factor-BB alone. Cartilage was present only in sites treated with insulin-like growth factor-I alone. These results suggest that the combination of platelet-derived growth factor-BB and insulin-like growth factor-I stimulates bone formation in wounds in long bones of adult animals and that these growth factors act via different pathways during the repair process.     

Proliferating and differentiating effects of three different growth factors on pluripotent mesenchymal cells and osteoblast like cells

Growth factors are in clinical use to stimulate bone growth and regeneration. BMP-2 is used in long bone and spinal surgery, PDGFbb for the treatment of periodontal defects and children with growth hormone receptor deficiency are treated with IGF-I.     

Improvement of fracture healing by systemic administration of growth hormone and local application of insulin-like growth factor-1 and transforming growth factor-beta1

Fracture healing is influenced by numerous hormones, growth factors, and cytokines. The systemic administration of growth hormone (GH) has shown to accelerate bone regeneration. Local application of growth factors, such as insulin-like growth factor-1 (IGF-1) and transforming growth factor-beta-1 (TGF-beta1), are known to stimulate bone metabolism. Until now, the exact local and systemic mechanisms that lead to improved bone regeneration remain unclear. In addition, the effect of systemic administration of GH as compared with locally delivered growth factors on fracture healing in rats is not known. A midshaft fracture of the right tibia of 5-month-old female Sprague-Dawley rats (n = 80) was intramedullary stabilized with IGF-1 and TGF-beta1 coated vs. uncoated titanium K-wires. The growth factors were incorporated in a poly(D,L-lactide) (PDLLA) coating and released continuously throughout the experiment. Recombinant species-specific (rat) GH was applied systemically (2 mg/kg body weight) by daily subcutaneous injection and compared with a placebo group. The healing process was radiologically monitored. Twenty-eight days after fracture biomechanical torsional testing was performed. The consolidation and callus composition, including quantification of cartilage and mineralized tissue, was traced in histomorphometrical investigations using an image analysis system. Both methods, the systemic administration of GH and the local application of growth factors, showed significant biomechanical and histological effects on fracture healing. The local growth factor application showed a stronger effect on fracture healing than the systemic GH injection. The combined application of both methods did not accelerate the effect on bone healing compared with the single application. It is therefore concluded that combining local and systemic stimulating methods does not provide further additive effects with regard to fracture healing.