Matrices and scaffolds for delivery of bioactive molecules in bone and cartilage tissue engineering

Regeneration of bone and cartilage defects can be accelerated by localized delivery of appropriate growth factors incorporated within biodegradable carriers. The carrier essentially allows the impregnated growth factor to release at a desirable rate and concentration, and to linger at injury sites for a sufficient time to recruit progenitors and stimulate tissue healing processes. In addition, the carrier can be formulated to have particular structure to facilitate cellular infiltration and growth. In this review, we present a summary of growth factor delivery carrier systems for bone and cartilage tissue engineering. Firstly, we describe a list of growth factors implicated in repair and regeneration of bone and cartilage by addressing their biological effects at different stages of the healing process. General requirements for localized growth factor delivery carriers are then discussed. We also provide selective examples of material types (natural and synthetic polymers, inorganic materials, and their composites) and fabricated forms of the carrier (porous scaffolds, microparticles, and hydrogels), highlighting the dose-dependent efficacy, release kinetics, animal models, and restored tissue types. Extensive discussion on issues involving currently investigated carriers for bone and cartilage tissue engineering approaches may illustrate future paths toward the development of an ideal growth factor delivery system.     

Combined gene and stem cell therapy for cutaneous wound healing

In current medical practice, wound therapy remains a clinical challenge and much effort has been focused on the development of novel therapeutic approaches for wound treatment. Gene therapy, initially developed for treatment of congenital defects, represents a promising option for enhancing wound repair. In order to accelerate wound closure, genes encoding for growth factors or cytokines have shown the most potential. The majority of gene delivery systems are based on viral transfection, naked DNA application, high pressure injection, and liposomal vectors. Besides advances stemming from breakthroughs in recombinant growth factors and bioengineered skin, there has been a significant increase in the understanding of stem cell biology in the field of cutaneous wound healing. A variety of sources, such as bone marrow, umbilical cord blood, adipose tissue and skin/hair follicles, have been utilized to isolate stem cells and to modulate the healing response of acute and chronic wounds. Recent data have demonstrated the feasibility of autologous adult stem cell therapy in cutaneous repair and regeneration. Very recently, stem cell based skin engineering in conjunction with gene recombination, in which the stem cells act as both the seed cells and the vehicle for gene delivery to the wound site, represents the most attractive field for generating a regenerative strategy for wound therapy. The aim of this article is to discuss the use and the potential of these novel technologies in order to improve wound healing capacities.     

Growth factor delivery for bone tissue engineering

Bone is a dynamic tissue that undergoes significant turnover during the life cycle of an individual. Despite having a significant regenerative capability, trauma and other pathological scenarios commonly require therapeutic intervention to facilitate the healing process. Bone tissue engineering, where cellular and biological processes at a site are deliberately manipulated for a therapeutic outcome, offers a viable option for the treatment of skeletal diseases. In this review paper, we aim to provide a brief synopsis of cellular and molecular basis of bone formation that are pertinent to current efforts of bone healing. Different approaches for engineering bone tissue were presented with special emphasis on the use of soluble (diffusible) therapeutic agents to accelerate bone healing. The latter agents have been used for both local bone repair (i.e. introduction of agents directly to a site of repair) as well as systemic bone regeneration (i.e. delivery for regeneration throughout the skeletal system). Critical drug delivery and targeting issues pertinent for each mode of bone regeneration are provided. In addition, future challenges and opportunities in bone tissue engineering are proposed from the authors' perspective.     

Spatial regulation of controlled bioactive factor delivery for bone tissue engineering

Limitations of current treatment options for critical size bone defects create a significant clinical need for tissue engineered bone strategies. This review describes how control over the spatiotemporal delivery of growth factors, nucleic acids, and drugs and small molecules may aid in recapitulating signals present in bone development and healing, regenerating interfaces of bone with other connective tissues, and enhancing vascularization of tissue engineered bone. State-of-the-art technologies used to create spatially controlled patterns of bioactive factors on the surfaces of materials, to build up 3D materials with patterns of signal presentation within their bulk, and to pattern bioactive factor delivery after scaffold fabrication are presented, highlighting their applications in bone tissue engineering. As these techniques improve in areas such as spatial resolution and speed of patterning, they will continue to grow in value as model systems for understanding cell responses to spatially regulated bioactive factor signal presentation in vitro, and as strategies to investigate the capacity of the defined spatial arrangement of these signals to drive bone regeneration in vivo.     

Regeneration of cartilage and bone by defined subsets of mesenchymal stromal cells--potential and pitfalls

Mesenchymal stromal cells, also referred to as mesenchymal stem cells, can be obtained from various tissues. Today the main source for isolation of mesenchymal stromal cells in mammals is the bone marrow. Mesenchymal stromal cells play an important role in tissue formation and organogenesis during embryonic development. Moreover, they provide the cellular and humoral basis for many processes of tissue regeneration and wound healing in infancy, adolescence and adulthood as well. There is increasing evidence that mesenchymal stromal cells from bone marrow and other sources including term placenta or adipose tissue are not a homogenous cell population. Only a restricted number of appropriate stem cells markers have been explored so far. But routine preparations of mesenchymal stromal cells contain phenotypically and functionally distinct subsets of stromal cells. Knowledge on the phenotypical characteristics and the functional consequences of such subsets will not only extend our understanding of stem cell biology, but might allow to develop improved regimen for regenerative medicine and wound healing and novel protocols for tissue engineering as well. In this review we will discuss novel strategies for regenerative medicine by specific selection or separation of subsets of mesenchymal stromal cells in the context of osteogenesis and bone regeneration. Mesenchymal stromal cells, which express the specific cell adhesion molecule CD146, also known as MCAM or MUC18, are prone for bone repair. Other cell surface proteins may allow the selection of chondrogenic, myogenic, adipogenic or other pre-determined subsets of mesenchymal stromal cells for improved regenerative applications as well.     

Localized delivery of growth factors for bone repair.

Delivery of growth factors for tissue (e.g. bone, cartilage) or cell repair (e.g. nerves) is about to gain important potential as a future therapeutic tool. Depending on the targeted cell type and its state of differentiation, growth factors can activate or regulate a variety of cellular functions. Therefore, strictly localized delivery regimens at well-defined kinetics appear to be logical prerequisites to assure safe and efficacious therapeutic use of such factors and avoid unwanted side effects and toxicity, a major hurdle in the clinical development of growth factor therapies so far. This review summarizes various approaches for localized growth factor delivery as focused on bone repair. Similar considerations may apply to other growth factors and therapeutic indications. Considering the vast number of preclinical studies reported in the area of growth factor-assisted bone repair, it surprises though that only two medical products for bone repair have so far been commercialized, both consisting of a collagen matrix impregnated with a bone morphogenetic protein. The marked diversity of the reported growth factors, delivery concepts and not yet standardized animal models adds to the complexity to learn from past preclinical studies presented in the literature. Nonetheless, it is now firmly established from the available information that the type, dose and delivery kinetics of growth factors all play a decisive role for the therapeutic success of any such approach. Very likely, all of these parameters have to be adapted and optimized for each animal model or clinical case. In the future, systems for localized growth factor delivery thus need to be designed in such a way that their modular components are readily adaptable to the individual pathology. To make such customized systems feasible, close cooperative networks of biomedical and biomaterials engineers, pharmaceutical scientists, chemists, biologists and clinicians need to be established.     

Biomaterial delivery of morphogens to mimic the natural healing cascade in bone

Complications in treatment of large bone defects using bone grafting still remain. Our understanding of the endogenous bone regeneration cascade has inspired the exploration of a wide variety of growth factors (GFs) in an effort to mimic the natural signaling that controls bone healing. Biomaterial-based delivery of single exogenous GFs has shown therapeutic efficacy, and this likely relates to its ability to recruit and promote replication of cells involved in tissue development and the healing process. However, as the natural bone healing cascade involves the action of multiple factors, each acting in a specific spatiotemporal pattern, strategies aiming to mimic the critical aspects of this process will likely benefit from the usage of multiple therapeutic agents. This article reviews the current status of approaches to deliver single GFs, as well as ongoing efforts to develop sophisticated delivery platforms to deliver multiple lineage-directing morphogens (multiple GFs) during bone healing.     

Biomaterial strategies for engineering implants for enhanced osseointegration and bone repair

Bone tissue has a remarkable ability to regenerate and heal itself. However, large bone defects and complex fractures still present a significant challenge to the medical community. Current treatments center on metal implants for structural and mechanical support and auto- or allo-grafts to substitute long bone defects. Metal implants are associated with several complications such as implant loosening and infections. Bone grafts suffer from donor site morbidity, reduced bioactivity, and risk of pathogen transmission. Surgical implants can be modified to provide vital biological cues, growth factors and cells in order to improve osseointegration and repair of bone defects. Here we review strategies and technologies to engineer metal surfaces to promote osseointegration with the host tissue. We also discuss strategies for modifying implants for cell adhesion and bone growth via integrin signaling and growth factor and cytokine delivery for bone defect repair.     

Demineralized bone matrix as a vehicle for delivering endogenous and exogenous therapeutics in bone repair

As a unique human bone extract approved for implant use, demineralized bone matrix (DBM) retains substantial amounts of endogenous osteoconductive and osteoinductive proteins. Commercial preparations of DBM represent a clinically accessible, familiar, widely used and degradable bone-filling device, available in composite solid, strip/piece, and semi-solid paste forms. Surgically placed and/or injected, DBM releases its constituent compounds to bone sites with some evidence for inducing new bone formation and accelerating healing. Significantly, DBM also has preclinical history as a drug carrier by direct loading and delivery of several important classes of therapeutics. Exogenous bioactive agents, including small molecule drugs, protein and peptide drugs, nucleic acid drugs and transgenes and therapeutic cells have been formulated within DBM and released to bone sites to enhance DBM's intrinsic biological activity. Local release of these agents from DBM directly to surgical sites in bone provides improved control of dosing and targeting of both endogenous and exogenous bioactivity in the context of bone healing using a clinically familiar product. Given DBM's long clinical track record and commercial accessibility in standard forms and sources, opportunities to formulate DBM as a versatile matrix to deliver therapeutic agents locally to bone sites in orthopedic repair and regenerative medicine contexts are attractive.     

Growth factor delivery for oral and periodontal tissue engineering

The treatment of oral and periodontal diseases and associated anomalies accounts for a significant proportion of the healthcare burden, with the manifestations of these conditions being functionally and psychologically debilitating. Growth factors are critical to the development, maturation, maintenance and repair of craniofacial tissues, as they establish an extracellular environment that is conducive to cell and tissue growth. Tissue-engineering principles aim to exploit these properties in the development of biomimetic materials that can provide an appropriate microenvironment for tissue development. These materials have been constructed into devices that can be used as vehicles for delivery of cells, growth factors and DNA. In this review, different mechanisms of drug delivery are addressed in the context of novel approaches to reconstruct and engineer oral- and tooth-supporting structures, namely the periodontium and alveolar bone.     

Biomimetic materials for tissue engineering

Tissue engineering and regenerative medicine is an exciting research area that aims at regenerative alternatives to harvested tissues for transplantation. Biomaterials play a pivotal role as scaffolds to provide three-dimensional templates and synthetic extracellular matrix environments for tissue regeneration. It is often beneficial for the scaffolds to mimic certain advantageous characteristics of the natural extracellular matrix, or developmental or wound healing programs. This article reviews current biomimetic materials approaches in tissue engineering. These include synthesis to achieve certain compositions or properties similar to those of the extracellular matrix, novel processing technologies to achieve structural features mimicking the extracellular matrix on various levels, approaches to emulate cell-extracellular matrix interactions, and biologic delivery strategies to recapitulate a signaling cascade or developmental/wound healing program. The article also provides examples of enhanced cellular/tissue functions and regenerative outcomes, demonstrating the excitement and significance of the biomimetic materials for tissue engineering and regeneration.     

Controlled release of drugs in electrosprayed nanoparticles for bone tissue engineering

Generating porous topographic substrates, by mimicking the native extracellular matrix (ECM) to promote the regeneration of damaged bone tissues, is a challenging process. Generally, scaffolds developed for bone tissue regeneration support bone cell growth and induce bone-forming cells by natural proteins and growth factors. Limitations are often associated with these approaches such as improper scaffold stability, and insufficient cell adhesion, proliferation, differentiation, and mineralization with less growth factor expression. Therefore, the use of engineered nanoparticles has been rapidly increasing in bone tissue engineering (BTE) applications. The electrospray technique is advantageous over other conventional methods as it generates nanomaterials of particle sizes in the micro/nanoscale range. The size and charge of the particles are controlled by regulating the polymer solution flow rate and electric voltage. The unique properties of nanoparticles such as large surface area-to-volume ratio, small size, and higher reactivity make them promising candidates in the field of biomedical engineering. These nanomaterials are extensively used as therapeutic agents and for drug delivery, mimicking ECM, and restoring and improving the functions of damaged organs. The controlled and sustained release of encapsulated drugs, proteins, vaccines, growth factors, cells, and nucleotides from nanoparticles has been well developed in nanomedicine. This review provides an insight into the preparation of nanoparticles by electrospraying technique and illustrates the use of nanoparticles in drug delivery for promoting bone tissue regeneration.     

The role of chemokines, cytokines and adhesion molecules in stem cell trafficking and homing

Although definition and mechanistic understanding of pro-angiogenic progenitor cells remains unsatisfactory, general agreement highlights their role in regenerative process following tissue injury and ischemia. Furthermore, stem-cell based therapy represents a hot topic of cardiovascular medicine. Recent studies provide new insights on the signalling pathways that modulate stem/progenitor cell mobilization from bone marrow, homing to ischemic area and participation in vascular remodelling and tissue healing. This review focuses on current knowledge and emerging concepts on stem cell/progenitor cell trafficking in relation to changes in surrounding environment and epigenetic modifications caused by risk factors and comorbidities.     

Co-culture systems for vascularization--learning from nature

The endothelial cell (EC) is practically ubiquitous in the human body and forms the inner cellular lining of the entire cardiovascular system. Following tissue injury, the microcirculation becomes the stage for both the inflammatory response and the subsequent healing reaction to restore physiological function to the damaged tissue. The advent of the multidisciplinary field of Regenerative Medicine (RegMed), of which Tissue Engineering (TE) and drug delivery using modern stimuli-responsive or interactive biomaterials are important components, has opened up new approaches to the acceleration of the healing response. A central and rate-limiting role in the latter is played by the process of vascularization or neovascularization, so that it is not surprising that in RegMed concepts have been developed for the drug- and gene-delivery of potent stimuli such as vascular-endothelial growth factor (VEGF) to promote neovessel development. However, not all of these novel materials can be tested in vivo, and in vitro co-culture model systems using human primary cells are being developed to pre-evaluate and determine which of the RegMed concepts exhibit the most promising potential for success after implantation. This review describes some of the growing number of in vitro co-cultures model systems that are being used to study cell-cell and cell-material interactions at the cellular and molecular levels to determine which materials are best suited to integrate into the host, promote a rapid vascularization and fit into the regenerative process without disturbing or slowing the normal healing steps.     

Drug and cell delivery for cardiac regeneration

The spectrum of ischaemic cardiomyopathy, encompassing acute myocardial infarction to congestive heart failure is a significant clinical issue in the modern era. This group of diseases is an enormous source of morbidity and mortality and underlies significant healthcare costs worldwide. Cardiac regenerative therapy, whereby pro-regenerative cells, drugs or growth factors are administered to damaged and ischaemic myocardium has demonstrated significant potential, especially preclinically. While some of these strategies have demonstrated a measure of success in clinical trials, tangible clinical translation has been slow. To date, the majority of clinical studies and a significant number of preclinical studies have utilised relatively simple delivery methods for regenerative therapeutics, such as simple systemic administration or local injection in saline carrier vehicles. Here, we review cardiac regenerative strategies with a particular focus on advanced delivery concepts as a potential means to enhance treatment efficacy and tolerability and ultimately, clinical translation. These include (i) delivery of therapeutic agents in biomaterial carriers, (ii) nanoparticulate encapsulation, (iii) multimodal therapeutic strategies and (iv) localised, minimally invasive delivery via percutaneous transcatheter systems.     

Topical astragaloside IV-releasing hydrogel improves healing of skin wounds in vivo

Topical delivery of therapeutic agents at the time of injury to accelerate skin repair and prevent the formation of scars during the wound healing process has received increasing attention and represents a novel regenerative and prophylactic strategy for wound treatment. The aim of this study was to invesigate, for the first time, the influence of topical astragaloside IV-releasing hydrogel on the wound repair and regeneration. Using the sodium alginate-gelatin as a hydrogel vehicle, the astragaloside IV was incorporated into the topical carrier and kept releasing with a sustained manner at the wound site. With the rat skin excision model, regulation of the astragaloside IV hydrogel on the wound repair and regeneration were investigated. It was found that the astragaloside IV hydrogel was effective in the skin wound repair, leading to a significant improvement on the wound closure, collagen synthesis and skin tensile strength recovery. Meanwhile, for the first time, that functions of astragaloside IV hydrogel in activating the skin appendages regeneration and increasing the transforming growth factor-β(1) (TGF-β(1)) level in serum were shown. Results of this study provided evidence for the alginate-gelatin hydrogel as efficient carrier for the topical delivery of bioactive molecules to the injured site. The astragaloside IV releasing hydrogel was shown a promising therapeutic formulation for wound healing, as well as its regenerative feature and underlying mechanism contribute to the skin regeneration were disclaimed.     

Bioresorbable and nonresorbable polymers for bone tissue engineering

In recent years, bone tissue engineering has emerged as one of the main research areas in the field of regenerative biomedicine. Frequency and relevance age-related diseases, such as healing and regeneration of bone tissues, are rising due to increasing life expectancy. Even though bone tissue has excellent self-regeneration ability, when bone defects exceed a critical size, impaired bone formation can occur and surgical intervention becomes mandatory. Bone tissue engineering represents an alternative approach to conventional bone transplants. The main aim of tissue engineering is to repair, regenerate or reconstruct damaged or degenerative tissue. This review presents an overview on the main materials, techniques and strategies in the field of bone tissue engineering. Whilst presenting some reviews recently published that deepen on each of the sections of the paper, this review article aims to present some of the most relevant advances, both in terms of new materials and strategies, currently being developed for bone repair and regeneration.     

The importance of drug delivery to optimize the effects of bone morphogenetic proteins during periodontal regeneration

Bone morphogenetic proteins (BMPs) include a large number of proteins belonging to the TGF-beta superfamily which are characterized by their ability to induce bone and cartilage formation. Since the isolation and purification of BMPs by recombinant technology, the effects of single BMPs can now be evaluated in animal models. Subcutanous placement of a single recombinant BMP, such as recombinant human (rh) BMP-2, in a rat ectopic assay shows recruitment of undifferentiated mesenchymal cells, cartilage formation, followed by replacement with bone, formation of its own bone marrow and physiological bone remodelling. The therapeutic use of recombinant BMPs in the treatment of periodontal disease (destruction of the tooth ligaments, surrounding bone and tooth cementum, the latter of which anchors the ligaments to the tooth surface from the adjacent tooth socket) has attracted considerable interest due to their potent ability to stimulate intramembranous bone formation without an endochondral intermediate. Their predictability in stimulating new bone may provide an alternative that has greater osteogenic potential than autogenous bone, other growth factors and bone substitutes. The biological processes and the potential role of growth factors involved in promoting regeneration are complicated by the involvement of different cell types each with their different growth rates and responses to various stimuli. The major cell types involved in periodontal regeneration include osteoblasts, cementoblasts and fibroblasts. Here, the formation of the new mineralized layers on the tooth and bone surfaces by cementoblasts and osteoblasts respectively are a prerequisite before periodontal ligament formation and attachment by fibroblasts can occur. In this regard, BMPs are likely candidates to stimulate periodontal regeneration because of their ability not only to promote osteogenesis but also to stimulate cementogenesis (new cementum formation). However, understanding when to manipulate each of the various cells differentiation pathway with the application of single or multiple doses of BMPs at the appropriate concentration is dependent upon a suitable delivery system that can be modified in order to optimize its effect during periodontal wound healing. Furthermore, treatment of intrabony periodontal defects with BMPs are likely to not only require appropriate temporal release of the agent, but also adaptation of a carrier that is robust enough to maintain its integrity around the coronal aspect of the root in order to provide space maintenance and support the mucoperiosteal flap. This review evaluates the effects of different delivery systems upon BMP-induced periodontal regeneration.     

Bone tissue engineering by gene delivery

Recombinant human bone morphogenetic protein-2 and -7 were recently granted United States Food and Drug Administration approval for select clinical applications in bone repair. While significant progress has been made in the delivery of recombinant osteogenic factor to promote bone healing, the short half-life and instability of the protein requires the delivery of milligram quantities of factor or multiple dosages. The potential of gene therapy for bone regeneration is the delivery of physiological levels of therapeutic protein using natural cellular mechanisms. Experimental investigations have demonstrated this approach uses lower dosages of factor to yield bone healing equivalent to that achieved via the administration of recombinant factor or use of bone grafts. The current states of gene delivery for bone tissue engineering applications and challenges to be met are presented in this review. Over the past couple of years, studies have continued to examine the delivery of the osteogenic factor bone morphogenetic protein using gene therapies. The importance of angiogenesis to bone formation has prompted the development of vascular endothelial growth factor gene expression systems for bone regeneration. Viral vectors, in combination with allograft bone, have been investigated to improve existing surgical care. Newly constructed vectors with reduced immunogenicity and regulated gene expression systems provide a greater degree of control over the timing and level of gene expression. Several advances have allowed bone tissue engineering by gene delivery to advance beyond serving as a potential treatment for isolated bone defects and fractures to a gene therapy approach for the treatment of genetic based bone diseases, such as osteogenesis imperfecta.