Tissue engineered scaffold utilizing the reinforced technique

Various types of biomaterials have been developed and utilized for the repair of damaged tissue and organs and the biocompatibility of these materials is of major concern. Tissue engineered scaffolds should provide adequate mechanical strength during the initial healing state, and the highly porous structure should provide an ideal environment for the migration and proliferation of cells; additionally, during surgical operations, the scaffolds should be able to be handled and sutured. Therefore, many researchers have designed reinforced scaffolds so as to address these issues. The structures described can be applied to the development of artificial ligaments, tendons, skin, bones, cartilage, and trachea.     

Optimized bone regeneration based on sustained release from three-dimensional fibrous PLGA/HAp composite scaffolds loaded with BMP-2

Contemporary treatment of critical bone defect remains a significant challenge in the field of orthopedic surgery. Engineered biomaterials combined with growth factors have emerged as a new treatment alternative in bone repair and regeneration. Our approach is to encapsulate bone morphogenetic protein-2 (BMP-2) into a polymeric matrix in different ways and characterize their individual performance in a nude mouse model. The main objective of this study is to examine whether the PLGA/HAp composite fibrous scaffolds loaded with BMP-2 through electrospinning can improve bone regeneration. The hypothesis is that different loading methods of BMP-2 and different HAp contents in scaffolds can alternate the release profiles of BMP-2 in vivo, therefore modify the performance of scaffolds in bone regeneration. Firstly, mechanical strength of scaffolds and HAp nanoparticles distribution in scaffolds were investigated. Secondly, nude mice experiments extended to 6 weeks were carried out to test the in vivo performance of these scaffolds, in which measurements, like serum BMP-2 concentration, ALP activity, X-ray qualification, and H&E/IHC tissue staining were utilized to monitor the growth of new bone and the changes of the corresponding biochemical parameters. The results showed that the PLGA/HAp composite scaffolds developed in this study exhibited good morphology/mechanical strength and HAp nanoparticles were homogeneously dispersed inside PLGA matrix. Results from the animal experiments indicate that the bioactivity of BMP-2 released from the fibrous PLGA/HAp composite scaffolds is well maintained, which further improves the formation of new bone and the healing of segmental defects in vivo. It is concluded that BMP-2 loaded PLGA/HAp composite scaffolds are promising for bone healing.     

Exosomes from tendon stem cells promote injury tendon healing through balancing synthesis and degradation of the tendon extracellular matrix

Tendon injuries are common musculoskeletal system disorders in clinical, but the regeneration ability of tendon is limited. Tendon stem cells (TSCs) have shown promising effect on tissue engineering and been used for the treatment of tendon injury. Exosomes that serve as genetic information carriers have been implicated in many diseases and physiological processes, but effect of exosomes from TSCs on tendon injury repair is unclear. The aim of this study is to make clear that the effect of exosomes from TSCs on tendon injury healing. Exosomes were harvested from conditioned culture media of TSCs by a sequential centrifugation process. Rat Achilles tendon tendinopathy model was established by collagenase‐I injection. This was followed by intra‐Achilles‐tendon injection with TSCs or exosomes. Tendon healing and matrix degradation were evaluated by histology analysis and biomechanical test at the post‐injury 5 weeks. In vitro, TSCs treated with interleukin 1 beta were added by conditioned medium including exosomes or not, or by exosomes or not. Tendon matrix related markers and tenogenesis related markers were measured by immunostaining and western blot. We found that TSCs injection and exosomes injection significantly decreased matrix metalloproteinases (MMP)‐3 expression, increased expression of tissue inhibitor of metalloproteinase‐3 (TIMP‐3) and Col‐1a1, and increased biomechanical properties of the ultimate stress and maximum loading. In vitro, conditioned medium with exosomes and exosomes also significantly decreased MMP‐3, and increased expression of tenomodulin, Col‐1a1 and TIMP‐3. Exosomes from TSCs could be an ideal therapeutic strategy in tendon injury healing for its balancing tendon extracellular matrix and promoting the tenogenesis of TSCs.     

Bone regeneration and stem cells

This invited review covers research areas of central importance for orthopaedic and maxillofacial bone tissue repair, including normal fracture healing and healing problems, biomaterial scaffolds for tissue engineering, mesenchymal and foetal stem cells, effects of sex steroids on mesenchymal stem cells, use of platelet-rich plasma for tissue repair, osteogenesis and its molecular markers. A variety of cells in addition to stem cells, as well as advances in materials science to meet specific requirements for bone and soft tissue regeneration by addition of bioactive molecules, are discussed.     

Repair and regeneration of osteochondral defects in the articular joints

People suffering from pain due to osteoarthritic or rheumatoidal changes in the joints are still waiting for a better treatment. Although some studies have achieved success in repairing small cartilage defects, there is no widely accepted method for complete repair of osteochondral defects. Also joint replacements have not yet succeeded in replacing of natural cartilage without complications. Therefore, there is room for a new medical approach, which outperforms currently used methods. The aim of this study is to show potential of using a tissue engineering approach for regeneration of osteochondral defects. The critical review of currently used methods for treatment of osteochondral defects is also provided. In this study, two kinds of hybrid scaffolds developed in Hutmacher's group have been analysed. The first biphasic scaffold consists of fibrin and PCL. The fibrin serves as a cartilage phase while the porous PCL scaffold acts as the subchondral phase. The second system comprises of PCL and PCL-TCP. The scaffolds were fabricated via fused deposition modeling which is a rapid prototyping system. Bone marrow-derived mesenchymal cells were isolated from New Zealand White rabbits, cultured in vitro and seeded into the scaffolds. Bone regenerations of the subchondral phases were quantified via micro CT analysis and the results demonstrated the potential of the porous PCL and PCL-TCP scaffolds in promoting bone healing. Fibrin was found to be lacking in this aspect as it degrades rapidly. On the other hand, the porous PCL scaffold degrades slowly hence it provides an effective mechanical support. This study shows that in the field of cartilage repair or replacement, tissue engineering may have big impact in the future. In vivo bone and cartilage engineering via combining a novel composite, biphasic scaffold technology with a MSC has been shown a high potential in the knee defect regeneration in the animal models. However, the clinical application of tissue engineering requires the future research work due to several problems, such as scaffold design, cellular delivery and implantation strategies.     

Mechanotransduction of stem cells for tendon repair

Tendon is a mechanosensitive tissue that transmits force from muscle to bone. Physiological loading contributes to maintaining the homeostasis and adaptation of tendon, but aberrant loading may lead to injury or failed repair. It is shown that stem cells respond to mechanical loading and play an essential role in both acute and chronic injuries, as well as in tendon repair. In the process of mechanotransduction, mechanical loading is detected by mechanosensors that regulate cell differentiation and proliferation via several signaling pathways. In order to better understand the stem-cell response to mechanical stimulation and the potential mechanism of the tendon repair process, in this review, we summarize the source and role of endogenous and exogenous stem cells active in tendon repair, describe the mechanical response of stem cells, and finally, highlight the mechanotransduction process and underlying signaling pathways.     

Biomimesis and biomorphic transformations: New concepts applied to bone regeneration

In the last decades the activity of material scientists was more and more directed to the development of biomimetic scaffolds, able to drive and address cell activity towards proper differentiation and the repair of diseased human tissues. In case of bone, this requires the synthesis of three-dimensional constructs able to exchange chemical signals promoting osteogenesis and to progressively be resorbed during the formation and remodelling of new bone. Besides, particularly for the regeneration of extensive portions of bone, a morphological and mechanical biomimesis is also required, to allow cell colonization and formation of a proper vascularization tree. The healing of load-bearing bones also requires scaffolds with a hierarchically organized morphology, to provide improved biomechanical behaviour and allow a proper mechano-transduction of the mechanical stimuli down to the cell level. The present paper is an overview of the current technologies and devices developed in the last decade for the regeneration of bone tissue. In particular, novel biomimetic and biomorphic scaffolds, obtained by the controlled transformation of native ligneous structures, promise to adequately face the problem of obtaining complex hierarchical structures, not achievable otherwise by any currently existing manufacturing techniques.     

Biomimesis and biomorphic transformations: new concepts applied to bone regeneration

In the last decades the activity of material scientists was more and more directed to the development of biomimetic scaffolds, able to drive and address cell activity towards proper differentiation and the repair of diseased human tissues. In case of bone, this requires the synthesis of three-dimensional constructs able to exchange chemical signals promoting osteogenesis and to progressively be resorbed during the formation and remodelling of new bone. Besides, particularly for the regeneration of extensive portions of bone, a morphological and mechanical biomimesis is also required, to allow cell colonization and formation of a proper vascularization tree. The healing of load-bearing bones also requires scaffolds with a hierarchically organized morphology, to provide improved biomechanical behaviour and allow a proper mechano-transduction of the mechanical stimuli down to the cell level. The present paper is an overview of the current technologies and devices developed in the last decade for the regeneration of bone tissue. In particular, novel biomimetic and biomorphic scaffolds, obtained by the controlled transformation of native ligneous structures, promise to adequately face the problem of obtaining complex hierarchical structures, not achievable otherwise by any currently existing manufacturing techniques.     

Mechano-regulation of stem cell differentiation and tissue regeneration in osteochondral defects

Cartilage defects that penetrate the subchondral bone can undergo spontaneous repair through the formation of a fibrous or cartilaginous tissue mediated primarily by mesenchymal stem cells from the bone marrow. This tissue is biomechanically inferior to normal articular cartilage, and is often observed to degrade over time. Whether or not biomechanical factors control the type and quality of the repair tissue, and its subsequent degradation, have yet to be elucidated. In this paper, we hypothesise a relationship between the mechanical environment of mesenchymal stem cells and their subsequent dispersal, proliferation, differentiation and death. The mechano-regulation stimulus is hypothesised to be a function of strain and fluid flow; these quantities are calculated using biphasic poroelastic finite element analysis. A finite element model of an osteochondral defect in the knee was created, and used to simulate the spontaneous repair process. The model predicts bone formation through both endochondral and direct intramembranous ossification in the base of the defect, cartilage formation in the centre of the defect and fibrous tissue formation superficially. Greater amounts of fibrous tissue formation are predicted as the size of the defect is increased. Large strains are predicted within the fibrous tissue at the articular surface, resulting in significant cell apoptosis. This result leads to the conclusion that repair tissue degradation is initiated in the fibrous tissue that forms at the articular surface. The success of the mechano-regulation model in predicting many of the cellular events that occur during osteochondral defect healing suggest that in the future it could be used as a tool for optimising scaffolds for tissue engineering.     

Cytocentrifugation: a convenient and efficient method for seeding tendon-derived cells into monolayer cultures or 3-D tissue engineering scaffolds

Tendon and ligament injuries are very common, requiring some 200,000 reconstructions per year in the USA. Autografting can be used to repair these but donor tissue is limited and harvesting leads to morbidity at the graft sites. Tissue engineering has been used to grow simple tissues such as skin, cartilage and bone and due to their low vascularity and simple structure, tendons should be ideal candidates for such an approach. Scaffolds are essential for tissue engineering as they provide structure and signals that regulate growth. However, they present a physical barrier to cell seeding with the majority of the cells congregating at the scaffold surface. To address this we used centrifugation to enhance penetration of tendon-derived cells to the centres of 3-D scaffolds. The process had no apparent deleterious effects on the cells and both plating efficiency and cell distribution improved. After attachment the cells continued to proliferate and deposit a collagenous matrix. Scaffold penetration was investigated using layers of Azowipes allowing the separation and examination of individual leaves. At relatively low g-forces, cells penetrated a stack of 6 Azowipes leaving cells attached to each leaf. These data suggest that cytocentrifugation improves the penetration and homogeneity of tendon derived cells in 3-D and monolayer cultures.     

Informing tendon tissue engineering with embryonic development

Tendon is a strong connective tissue that transduces muscle-generated forces into skeletal motion. In fulfilling this role, tendons are subjected to repeated mechanical loading and high stress, which may result in injury. Tissue engineering with stem cells offers the potential to replace injured/damaged tissue with healthy, new living tissue. Critical to tendon tissue engineering is the induction and guidance of stem cells towards the tendon phenotype. Typical strategies have relied on adult tissue homeostatic and healing factors to influence stem cell differentiation, but have yet to achieve tissue regeneration. A novel paradigm is to use embryonic developmental factors as cues to promote tendon regeneration. Embryonic tendon progenitor cell differentiation in vivo is regulated by a combination of mechanical and chemical factors. We propose that these cues will guide stem cells to recapitulate critical aspects of tenogenesis and effectively direct the cells to differentiate and regenerate new tendon. Here, we review recent efforts to identify mechanical and chemical factors of embryonic tendon development to guide stem/progenitor cell differentiation toward new tendon formation, and discuss the role this work may have in the future of tendon tissue engineering.     

Gene therapy for tissue regeneration

Tissue repair and regeneration are the normal biological responses of many different tissues in the body to injury. During the healing process, profound changes occur in cell composition and extracellular matrix (ECM) formation. Fibroblasts and equivalent reparative cells migrate to the wounded area and subsequently proliferate. These cells and reparative cells from the surrounding tissue are responsible for the rapid repair which results in tissue regeneration. Growth factors, one of which is transforming growth factor-beta (TGF-beta), stimulate fibroblasts and smooth muscle cells to proliferate and synthesize ECM proteins. This process of early repair provides a rapid way to restore new tissue and mechanical integrity. This early tissue repair process is normally followed by involution, which requires the production and activation of proteases, tissue maturation and remodeling, reorganization and finally regeneration. Alternately, failure to replace the critical components of the ECM, including elastin and basement membrane, results in abnormal regeneration of the epithelial cell layer. Although remodeling should occur during healing, provisional repair may be followed by excessive synthesis and deposition of collagen, which results in irreversible fibrosis and scarring. This excessive fibrosis which occurs in aberrant healing is at least in part mediated by persistent TGF-beta. Because of the central role of collagen in the wound healing process, the pharmacological control of collagen synthesis has been of paramount importance as a possible way to abrogate aberrant healing and prevent irreversible fibrosis. Fibrosis is an abnormal response to tissue injury.     

In vitro Response of the Bone Marrow-Derived Mesenchymal Stem Cells Seeded in a Type-I Collagen-Glycosaminoglycan Scaffold for Skin Wound Repair Under the Mechanical Loading Condition

In order to achieve successful wound repair by regenerative tissue engineering using mesenchymal stem cells (MSCs), it is important to understand the response of stem cells in the scaffold matrix to mechanical stress.
To investigate the clinical effects of mechanical stress on the behavior of cells in scaffolds, bone marrow-derived mesenchymal stem cells (MSCs) were grown on a type-I collagen-glycosaminoglycan (GAG) scaffold matrix for one week under cyclic stretching loading conditions.
The porous collagen-GAG scaffold matrix for skin wound repair was prepared, the harvested canine MSCs were seeded on the scaffold, and cultured under three kinds of cyclic stretching loading conditions ( 0%: control, 5% strain, 15% strain ). After 7 days incubation, MSCs were evaluated histologically and immunohistochemically regarding the proliferation and differentiation.
Cultured MSCs in the high strain (15% strain) group showed activea-smooth muscle actin (α-SMA) expression and poor differentiation into type-I collagen-positive cells, whereas enhanced differentiation into type-I collagen positive cells and a lack ofa-SMA expression where shown in the lower stress (5% strain) group. These results suggest that mechanical stress may affect the proliferation and differentiation of stem cells, and subsequently the wound healing process, through attachment interactions between the stem cells and scaffold matrix. Our findings provide an additional consideration for clinical treatment of wound repair using regenerative tissue engineering.     

Stem cell therapy in the management of shoulder rotator cuff disorders

Rotator cuff tears are frequent shoulder problems that are usually dealt with surgical repair. Despite improved surgical techniques, the tendon-to-bone healing rate is unsatisfactory due to difficulties in restoring the delicate transitional tissue between bone and tendon. It is essential to understand the molecular mechanisms that determine this failure. The study of the molecular environment during embryogenesis and during normal healing after injury is key in devising strategies to get a successful repair. Mesenchymal stem cells (MSC) can differentiate into different mesodermal tissues and have a strong paracrine, anti-inflammatory, immunoregulatory and angiogenic potential. Stem cell therapy is thus a potentially effective therapy to enhance rotator cuff healing. Promising results have been reported with the use of autologous MSC of different origins in animal studies: they have shown to have better healing properties, increasing the amount of fibrocartilage formation and improving the orientation of fibrocartilage fibers with less immunologic response and reduced lymphocyte infiltration. All these changes lead to an increase in biomechanical strength. However, animal research is still inconclusive and more experimental studies are needed before human application. Future directions include expanded stem cell therapy in combination with growth factors or different scaffolds as well as new stem cell types and gene therapy.     

In situ cell–matrix mechanics in tendon fascicles and seeded collagen gels: implications for the multiscale design of biomaterials

Designing biomaterials to mimic and function within the complex mechanobiological conditions of connective tissues requires a detailed understanding of the micromechanical environment of the cell. The objective of our study was to measure the in situ cell–matrix strains from applied tension in both tendon fascicles and cell-seeded type I collagen scaffolds using laser scanning confocal microscopy techniques. Tendon fascicles and collagen gels were fluorescently labelled to simultaneously visualise the extracellular matrix and cell nuclei under applied tensile strains of 5%. There were significant differences observed in the micromechanics at the cell–matrix scale suggesting that the type I collagen scaffold did not replicate the pattern of native tendon strains. In particular, although the overall in situ tensile strains in the matrix were quite similar (～2.5%) between the tendon fascicles and the collagen scaffolds, there were significant differences at the cell–matrix boundary with visible shear across cell nuclei of >1 μm measured in native tendon which was not observed at all in the collagen scaffolds. Similarly, there was significant non-uniformity of intercellular strains with relative sliding observed between cell rows in tendon which again was not observed in the collagen scaffolds where the strain environment was much more uniform. If the native micromechanical environment is not replicated in biomaterial scaffolds, then the cells may receive incorrect or mixed mechanical signals which could affect their biosynthetic response to mechanical load in tissue engineering applications. This study highlights the importance of considering the microscale mechanics in the design of biomaterial scaffolds and the need to incorporate such features in computational models of connective tissues.     

Dynamic compression promotes proliferation and neovascular networks of endothelial progenitor cells in demineralized bone matrix scaffold seed

Neovascularization is required for bone formation and successful fracture healing. In the process of neovascularization, endothelial progenitor cells (EPCs) play an important role and finish vascular repair through reendothelialization to promote successful fracture healing. In this study, we found that dynamic compression can promote the proliferation and capillary-like tube formation of EPCs in the demineralized bone matrix (DBM) scaffold seed. EPCs isolated from the bone marrow of rats have been cultured in DBM scaffolds before dynamic compression and then seeded in the DBM scaffolds under dynamic conditions. The cells/scaffold constructs were subjected to cyclic compression with 5% strain and at 1 Hz for 4 h/day for 7 consecutive days. By using MTT and real-time PCR, we found that dynamic compression can significantly induce the proliferation of EPCs in three-dimensional culture with an even distribution of cells onto DBM scaffolds. Both in vitro and in vivo, the tube formation assays in the scaffolds showed that the loaded EPCs formed significant tube-like structures. These findings suggest that dynamic compression promoted the vasculogenic activities of EPCs seeded in the scaffolds, which would benefit large bone defect tissue engineering.     

Early responses to mechanical load in tendon: role for calcium signaling, gap junctions and intercellular communication

Tendon and other connective tissue cells are subjected to diverse mechanical loads during daily activities. Thus, fluid flow, strain, shear and combinations of these stimuli activate mechanotransduction pathways that modulate tissue maintenance, repair and pathology. Early mechanotransduction events include calcium (Ca2+) signaling and intercellular communication. These responses are mediated through multiple mechanisms involving stretch-activated channels, voltage-activated channels such as Ca(v)1, purinoceptors, adrenoceptors, ryanodine receptor-mediated Ca2+ release, gap junctions and connexin hemichannels. Calcium, diacylglycerol, inositol (1,4,5)-trisphosphate, nucleotides and nucleosides play intracellular and/or extracellular signaling roles in these pathways. In addition, responses to mechanical loads in tendon cells vary among species, tendon type, anatomic location, loading conditions and other factors. This review includes a synopsis of the immediate responses to mechanical loading in connective tissue cells, particularly tenocytes. These responses involve Ca2+ signaling, gap junctions and intercellular communication.