Effects of three-dimensional scaffolds on cell organization and tissue development

Tissue engineering scaffolds play a critical role in regulating the reconstructed human tissue development. Various types of scaffolds have been developed in recent years, including fibrous matrix and foam-like scaffolds. The design of scaffold materials has been investigated extensively. However, the design of physical structure of the scaffold, especially fibrous matrices, has not received much attention. This paper compares the different characteristics of fibrous and foam-like scaffolds, and reviews regulatory roles of important scaffold properties, including surface geometry, scaffold configuration, pore structure, mechanical property and bioactivity. Tissue regeneration, cell organization, proliferation and differentiation under different microstructures were evaluated. The importance of proper scaffold selection and design is further discussed with the examples of bone tissue engineering and stem cell tissue engineering. This review addresses the importance of scaffold microstructure and provides insights in designing appropriate scaffold structure for different applications of tissue engineering.     

Permeability analysis of scaffolds for bone tissue engineering

Porous artificial bone substitutes, especially bone scaffolds coupled with osteobiologics, have been developed as an alternative to the traditional bone grafts. The bone scaffold should have a set of properties to provide mechanical support and simultaneously promote tissue regeneration. Among these properties, scaffold permeability is a determinant factor as it plays a major role in the ability for cells to penetrate the porous media and for nutrients to diffuse. Thus, the aim of this work is to characterize the permeability of the scaffold microstructure, using both computational and experimental methods. Computationally, permeability was estimated by homogenization methods applied to the problem of a fluid flow through a porous media. These homogenized permeability properties are compared with those obtained experimentally. For this purpose a simple experimental setup was used to test scaffolds built using Solid Free Form techniques. The obtained results show a linear correlation between the computational and the experimental permeability. Also, this study showed that permeability encompasses the influence of both porosity and pore size on mass transport, thus indicating its importance as a design parameter. This work indicates that the mathematical approach used to determine permeability may be useful as a scaffold design tool.     

Geometry Design Optimization of Functionally Graded Scaffolds for Bone Tissue Engineering: A Mechanobiological Approach

Functionally Graded Scaffolds (FGSs) are porous biomaterials where porosity changes in space with a specific gradient. In spite of their wide use in bone tissue engineering, possible models that relate the scaffold gradient to the mechanical and biological requirements for the regeneration of the bony tissue are currently missing. In this study we attempt to bridge the gap by developing a mechanobiology-based optimization algorithm aimed to determine the optimal graded porosity distribution in FGSs. The algorithm combines the parametric finite element model of a FGS, a computational mechano-regulation model and a numerical optimization routine. For assigned boundary and loading conditions, the algorithm builds iteratively different scaffold geometry configurations with different porosity distributions until the best microstructure geometry is reached, i.e. the geometry that allows the amount of bone formation to be maximized. We tested different porosity distribution laws, loading conditions and scaffold Young’s modulus values. For each combination of these variables, the explicit equation of the porosity distribution law–i.e the law that describes the pore dimensions in function of the spatial coordinates–was determined that allows the highest amounts of bone to be generated. The results show that the loading conditions affect significantly the optimal porosity distribution. For a pure compression loading, it was found that the pore dimensions are almost constant throughout the entire scaffold and using a FGS allows the formation of amounts of bone slightly larger than those obtainable with a homogeneous porosity scaffold. For a pure shear loading, instead, FGSs allow to significantly increase the bone formation compared to a homogeneous porosity scaffolds. Although experimental data is still necessary to properly relate the mechanical/biological environment to the scaffold microstructure, this model represents an important step towards optimizing geometry of functionally graded scaffolds based on mechanobiological criteria.     

A Mechanobiology-based Algorithm to Optimize the Microstructure Geometry of Bone Tissue Scaffolds

Complexity of scaffold geometries and biological mechanisms involved in the bone generation process make the design of scaffolds a quite challenging task. The most common approaches utilized in bone tissue engineering require costly protocols and time-consuming experiments. In this study we present an algorithm that, combining parametric finite element models of scaffolds with numerical optimization methods and a computational mechano-regulation model, is able to predict the optimal scaffold microstructure. The scaffold geometrical parameters are perturbed until the best geometry that allows the largest amounts of bone to be generated, is reached. We study the effects of the following factors: (1) the shape of the pores; (2) their spatial distribution; (3) the number of pores per unit area. The optimal dimensions of the pores have been determined for different values of scaffold Young''s modulus and compression loading acting on the scaffold upper surface.Pores with rectangular section were predicted to lead to the formation of larger amounts of bone compared to square section pores; similarly, elliptic pores were predicted to allow the generation of greater amounts of bone compared to circular pores. The number of pores per unit area appears to have rather negligible effects on the bone regeneration process. Finally, the algorithm predicts that for increasing loads, increasing values of the scaffold Young''s modulus are preferable.The results shown in the article represent a proof-of-principle demonstration of the possibility to optimize the scaffold microstructure geometry based on mechanobiological criteria.     

A mathematical model for bone tissue regeneration inside a specific type of scaffold

Bone tissue regeneration using scaffolds is receiving an increasing interest in orthopedic surgery and tissue engineering applications. In this study, we present the geometrical characterization of a specific family of scaffolds based on a face cubic centered (FCC) arrangement of empty pores leading to analytical formulae of porosity and specific surface. The effective behavior of those scaffolds, in terms of mechanical properties and permeability, is evaluated through the asymptotic homogenization theory applied to a representative volume element identified with the unit cell FCC. Bone growth into the scaffold is estimated by means of a phenomenological model that considers a macroscopic effective stress as the mechanical stimulus that regulates bone formation. Cell migration within the scaffold is modeled as a diffusion process based on Fick's law which allows us to estimate the cell invasion into the scaffold microstructure. The proposed model considers that bone growth velocity is proportional to the concentration of cells and regulated by the mechanical stimulus. This model allows us to explore what happens within the scaffold, the surrounding bone and their interaction. The mathematical model has been numerically implemented and qualitatively compared with previous experimental results found in the literature for a scaffold implanted in the femoral condyle of a rabbit. Specifically, the model predicts around 19 and 23% of bone regeneration for non-grafted and grafted scaffolds, respectively, both with an initial porosity of 76%.     

Composite scaffolds for cartilage tissue engineering

Tissue engineering remains a promising therapeutic strategy for the repair or regeneration of diseased or damaged tissues. Previous approaches have typically focused on combining cells and bioactive molecules (e.g., growth factors, cytokines and DNA fragments) with a biomaterial scaffold that functions as a template to control the geometry of the newly formed tissue, while facilitating the attachment, proliferation, and differentiation of embedded cells. Biomaterial scaffolds also play a crucial role in determining the functional properties of engineered tissues, including biomechanical characteristics such as inhomogeneity, anisotropy, nonlinearity or viscoelasticity. While single-phase, homogeneous materials have been used extensively to create numerous types of tissue constructs, there continue to be significant challenges in the development of scaffolds that can provide the functional properties of load-bearing tissues such as articular cartilage. In an attempt to create more complex scaffolds that promote the regeneration of functional engineered tissues, composite scaffolds comprising two or more distinct materials have been developed. This paper reviews various studies on the development and testing of composite scaffolds for the tissue engineering of articular cartilage, using techniques such as embedded fibers and textiles for reinforcement, embedded solid structures, multi-layered designs, or three-dimensionally woven composite materials. In many cases, the use of composite scaffolds can provide unique biomechanical and biological properties for the development of functional tissue engineering scaffolds.     

Infection and tissue engineering in segmental bone defects--a mini review

As tissue engineering becomes more of a clinical reality through the ongoing bench to bedside transition, research in this field must focus on addressing relevant clinical situations. Although most in vivo work in the area of bone tissue engineering focuses on bone regeneration within sterile, surgically created defects, there is a growing need for the investigation of bone tissue engineering approaches within contaminated or scarred wound beds, such as those that may be encountered following traumatic injury or during delayed reconstruction/regeneration. Significant work has been performed in the area of local drug delivery via biomaterial carriers, but there is little intersection in the available literature between antibiotic delivery and tissue regeneration. In this review, we examine recent advances in segmental bone defect animal models, bone tissue engineering, and drug delivery with the goal of identifying promising approaches and areas needing further investigation towards developing both a better understanding of and new tissue engineering approaches for addressing infection control while simultaneously initiating bone regeneration.     

The effects of gelatin,fibrin-platelet glue and their combination on healing of the experimental critical bone defect in a rat model: radiological,histological, scanning ultrastructural and biomechanical evaluation

Fibrin-platelet glue (FPG) is a blood derivative, in which platelets and fibrinogen are concentrated in a small plasma volume, by differential centrifugation and precipitation. It can form a three-dimensional and biocompatible fibrin scaffold with a myriad of growth factors and proteins that are released progressively to the local environment and contribute to the accelerated postoperative bone healing. Gelatin (Gel) is a derivative of collagen and can promote cell adhesion and proliferation due to its unique sequence of amino acids, so it is suitable for bone tissue applications. This study examined the effects of Gel, FPG and their combinations as bone scaffold on the healing of surgically created critical-size defects in rat radius. Fifty critical size defects of 5 mm long were bilaterally created in the radial diaphysis of 25 rats. The animals were randomly divided into five equal groups as empty defect, autograft, Gel, FPG and Gel–FPG groups (n = 10 in each group). Radiographs of each forelimb were taken postoperatively on the 1st day and then at the 28th and 56th days post injury to evaluate bone formation, union and remodeling of the defect. After 56 days, the rats were euthanized and their harvested healing bone samples were evaluated by histopathology, scanning electron microscopy (SEM) and biomechanical testing. The results of present study showed that the Gel alone did not significantly affect bone healing and regeneration; however, the Gel treated defects promoted healing more than those that were left untreated (negative control). Furthermore, the FPG-enhanced grafts provided a good scaffold containing numerous growth factors for proliferation of osteoinduction and was effective in improving the structural and functional properties of the newly formed bone more than that of the untreated and also the Gel treated groups. Incorporation of Gel into the FPG scaffold improved healing potential of the FPG scaffold; however, it was still inferior to the autograft (positive control). Although the Gel–FPG scaffolds had best effectiveness during bone regeneration, it still needs to be further enhanced by incorporation of the ceramic and osteoinductive biomaterials.     

Finite element method (FEM), mechanobiology and biomimetic scaffolds in bone tissue engineering

Techniques of bone reconstructive surgery are largely based on conventional, non-cell-based therapies that rely on the use of durable materials from outside the patient's body. In contrast to conventional materials, bone tissue engineering is an interdisciplinary field that applies the principles of engineering and life sciences towards the development of biological substitutes that restore, maintain, or improve bone tissue function. Bone tissue engineering has led to great expectations for clinical surgery or various diseases that cannot be solved with traditional devices. For example, critical-sized defects in bone, whether induced by primary tumor resection, trauma, or selective surgery have in many cases presented insurmountable challenges to the current gold standard treatment for bone repair. The primary purpose of bone tissue engineering is to apply engineering principles to incite and promote the natural healing process of bone which does not occur in critical-sized defects. The total market for bone tissue regeneration and repair was valued at $1.1 billion in 2007 and is projected to increase to nearly $1.6 billion by 2014.Usually, temporary biomimetic scaffolds are utilized for accommodating cell growth and bone tissue genesis. The scaffold has to promote biological processes such as the production of extra-cellular matrix and vascularisation, furthermore the scaffold has to withstand the mechanical loads acting on it and to transfer them to the natural tissues located in the vicinity. The design of a scaffold for the guided regeneration of a bony tissue requires a multidisciplinary approach. Finite element method and mechanobiology can be used in an integrated approach to find the optimal parameters governing bone scaffold performance.In this paper, a review of the studies that through a combined use of finite element method and mechano-regulation algorithms described the possible patterns of tissue differentiation in biomimetic scaffolds for bone tissue engineering is given. Firstly, the generalities of the finite element method of structural analysis are outlined; second, the issues related to the generation of a finite element model of a given anatomical site or of a bone scaffold are discussed; thirdly, the principles on which mechanobiology is based, the principal theories as well as the main applications of mechano-regulation models in bone tissue engineering are described; finally, the limitations of the mechanobiological models and the future perspectives are indicated.     

Scaffold-based bone engineering by using genetically modified cells

The first generation of clinically applied tissue engineering concepts in the area of skin, cartilage and bone marrow regeneration was based on the isolation, expansion and implantation of cells from the patient's own tissue. Although successful in selective treatments, tissue engineering needs to overcome major challenges to allow widespread clinical application with predictable outcomes. One challenge is to present the cells in a matrix to the implantation site to allow the cells to survive the wound healing contraction forces, tissue remodeling in certain tissues such as bone and biomechanical loading. Hence, several tissue engineering strategies focus on the development of load-bearing scaffold/cell constructs. From a cell source point of view, bone engineers face challenges to isolate and expand cells with the highest potential to form osseous tissue along with harvesting tissue without extensive donor site morbidity. A major hurdle to tissue engineering is de-differentiation and limited ability to control cell phenotype following in vitro expansion. Due to early successes with genetic engineering, bone tissue engineers have used different strategies to genetically alter various types of mesenchymal cells to enhance the mineralization capacity of tissue-engineered scaffold/cell constructs. Although the development of multi-component scaffold/osteogenic cell constructs requires a combination of interdisciplinary research strategies, the following review is limited to describe the general aspects of bone engineering and to present overall directions of technology platforms, which include a genetic engineering component. This paper reviews the most recent work in the field and discusses the concepts developed and executed by a collaborative effort of the multi-disciplinary teams of the two authors.     

A novel collagen scaffold supports human osteogenesis—applications for bone tissue engineering

Collagen glycosaminoglycan (CG) scaffolds have been clinically approved as an application for skin regeneration. The goal of this study has been to examine whether a CG scaffold is a suitable biomaterial for generating human bone tissue. Specifically, we have asked the following questions: (1) can the scaffold support human osteoblast growth and differentiation and (2) how might recombinant human transforming growth factor-beta (TGF-β₁) enhance long-term in vitro bone formation? We show human osteoblast attachment, infiltration and uniform distribution throughout the construct, reaching the centre within 14 days of seeding. We have identified the fully differentiated osteoblast phenotype categorised by the temporal expression of alkaline phosphatase, collagen type 1, osteonectin, bone sialo protein, biglycan and osteocalcin. Mineralised bone formation has been identified at 35 days post-seeding by using von Kossa and Alizarin S Red staining. Both gene expression and mineral staining suggest the benefit of introducing an initial high treatment of TGF-β₁ (10 ng/ml) followed by a low continuous treatment (0.2 ng/ml) to enhance human osteogenesis on the scaffold. Osteogenesis coincides with a reduction in scaffold size and shape (up to 70% that of original). A notable finding is core degradation at the centre of the tissue-engineered construct after 49 days of culture. This is not observed at earlier time points. Therefore, a maximum of 35 days in culture is appropriate for in vitro studies of these scaffolds. We conclude that the CG scaffold shows excellent potential as a biomaterial for human bone tissue engineering.     

Anterior Cruciate Ligament Reconstruction in a Rabbit Model Using Silk-Collagen Scaffold and Comparison with Autograft

The objective of the present study was to perform an in vivo assessment of a novel silk-collagen scaffold for anterior cruciate ligament (ACL) reconstruction. First, a silk-collagen scaffold was fabricated by combining sericin-extracted knitted silk fibroin mesh and type I collagen to mimic the components of the ligament. Scaffolds were electron-beam sterilized and rolled up to replace the ACL in 20 rabbits in the scaffold group, and autologous semitendinosus tendons were used to reconstruct the ACL in the autograft control group. At 4 and 16 weeks after surgery, grafts were retrieved and analyzed for neoligament regeneration and tendon-bone healing. To evaluate neoligament regeneration, H&E and immunohistochemical staining was performed, and to assess tendon-bone healing, micro-CT, biomechanical test, H&E and Russell-Movat pentachrome staining were performed. Cell infiltration increased over time in the scaffold group, and abundant fibroblast-like cells were found in the core of the scaffold graft at 16 weeks postoperatively. Tenascin-C was strongly positive in newly regenerated tissue at 4 and 16 weeks postoperatively in the scaffold group, similar to observations in the autograft group. Compared with the autograft group, tendon-bone healing was better in the scaffold group with trabecular bone growth into the scaffold. The results indicate that the silk-collagen scaffold has considerable potential for clinical application.     

State of the Art Technology for Bone Tissue Engineering and Drug Delivery

BackgroundCritical size bone defect and fracture unable to regenerate itself, inspire the origination and technological advancement in the field of bone tissue engineering (BTE). The strategies of bone tissue engineering are often classified into three groups: First, is a direct injection of cells into the tissue of interest; second is grafting of cell-scaffold constructs; and third is scaffold-based signaling molecules, drug delivery or both. Much research was available on the first two categories, still finding the structure and property of scaffold close towards the natural tissue is yet to achieve.Aim of the ReviewThe proposed mini review focus on ceramic biomaterials uses for bone regeneration and drug delivery. It covers the fabrication process of scaffold including conventional and non-conventional i.e. rapid prototyping approach along with it advantage. The use of scaffold for drug delivery and signaling molecules such as growth factor is an emerging field of research in tissue engineering.ConclusionThe biodegradable beads used as a local drug delivery system are ubiquitous in surgery to treat post-operative infections but does not play any role in tissue regeneration. The use of this clinically accepted drug delivery technique in bone regeneration is an alternative way for the treatment on several bone infections (especially osteomyelitis and arthritis associated with tuberculosis). It is predicted to be the future of organ replacement and treatment.     

Mechanical characterization of regenerated osseous tissue during callotasis and its related biological phenomenon

The mechanical characteristics of the regenerated osseous tissue and osteoblastic activities during callotasis were studied using Chinese mountain goat as the animal model. Open osteotomy of the left tibiae was done in 24 goats. Distraction started 6 days after the operation with the rate of 1 mm per day for 4 weeks. The bone regeneration was monitored with serial X-ray films. The tension generated during distraction was measured with the strain gauges mounted on the distraction apparatus. The osteoblastic activities were monitored by measuring plasma bone specific alkaline phosphatase activity. The results showed that the average lengthening was 22.9 +/- 2.8 mm in each animal. The newly formed osseous tissue becomes stiffer during the course of distraction lengthening. The continuous evolution of the tensile behaviour of the newly formed osseous tissue correlates with the plasma bone specific alkaline phosphatase activities. The radiological appearance of a physis like structure took place at 12 mm lengthening. Its appearance corresponds to the changes in the tensile behaviour as well as the biological activities of the osteoblasts and may serve as a useful radiological marker in monitoring the process of callotasis in clinical practice.     

A morphological study of regeneration of the goldfish elasmoid scales

Studies were carried out on the model of regeneration of mechanically removed elasmoid scales. Regenerating scales were morphologically analyzed using light and electron microscopy. It was found that the cells responsible for regeneration of the elasmoid scale plates could be classified as osteogenic elements. Little differentiated preosteoblasts were detected in the connective tissue of dermis on day 3 of regeneration, while partially calcified plates underlaid osteoblasts on day 7. The scale cover was fully restored on day 14 and it took two days for each bone plate to be formed. Osteocytes, fully differentiated osteogenic elements, were found in the deepest regions of newly formed scales.     

A morphological study of regeneration of the goldfish elasmoid scales

Studies were carried out on the model of regeneration of mechanically removed elasmoid scales. Regenerating scales were morphologically analyzed using light and electron microscopy. It was found that the cells responsible for regeneration of the elasmoid scale plates could be classified as osteogenic elements. Little differentiated preosteoblasts were detected in the connective tissue of dermis on day 3 of regeneration, while partially calcified plates underlay osteoblasts on day 7. The scale cover was fully restored on day 14 and it took two days for each bone plate to be formed. Osteocytes, fully differentiated osteogenic elements, were found in the deepest regions of newly formed scales.     

Magnetic hydroxyapatite bone substitutes to enhance tissue regeneration: evaluation in vitro using osteoblast-like cells and in vivo in a bone defect

In case of degenerative disease or lesion, bone tissue replacement and regeneration is an important clinical goal. In particular, nowadays, critical size defects rely on the engineering of scaffolds that are 3D structural supports, allowing cellular infiltration and subsequent integration with the native tissue. Several ceramic hydroxyapatite (HA) scaffolds with high porosity and good osteointegration have been developed in the past few decades but they have not solved completely the problems related to bone defects. In the present study we have developed a novel porous ceramic composite made of HA that incorporates magnetite at three different ratios: HA/Mgn 95/5, HA/Mgn 90/10 and HA/Mgn 50/50. The scaffolds, consolidated by sintering at high temperature in a controlled atmosphere, have been analysed in vitro using human osteoblast-like cells. Results indicate high biocompatibility, similar to a commercially available HA bone graft, with no negative effects arising from the presence of magnetite or by the use of a static magnetic field. HA/Mgn 90/10 was shown to enhance cell proliferation at the early stage. Moreover, it has been implanted in vivo in a critical size lesion of the rabbit condyle and a good level of histocompatibility was observed. Such results identify this scaffold as particularly relevant for bone tissue regeneration and open new perspectives for the application of a magnetic field in a clinical setting of bone replacement, either for magnetic scaffold fixation or magnetic drug delivery.     

One-year clinical study of NeuroRegen scaffold implantation following scar resection in complete chronic spinal cord injury patients

The objective of this clinical study was to assess the safety and feasibility of the collagen scaffold, Neuro Regen scaffold, one year after scar tissue resection and implantation. Scar tissue is a physical and chemical barrier that prevents neural regeneration. However, identification of scar tissue is still a major challenge. In this study, the nerve electrophysiology method was used to distinguish scar tissue from normal neural tissue, and then different lengths of scars ranging from 0.5–4.5 cm were surgically resected in five complete chronic spinal cord injury(SCI) patients. The NeuroR egen scaffold along with autologous bone marrow mononuclear cells(BMMCs), which have been proven to promote neural regeneration and SCI recovery in animal models, were transplanted into the gap in the spinal cord following scar tissue resection. No obvious adverse effects related to scar resection or Neuro Regen scaffold transplantation were observed immediately after surgery or at the 12-month follow-up. In addition, patients showed partially autonomic nervous function improvement, and the recovery of somatosensory evoked potentials(SSEP) from the lower limbs was also detected. The results indicate that scar resection and Neuro Regen scaffold transplantation could be a promising clinical approach to treating SCI.     

Mechanism of bone induction by KUSA/A1 cells using atelocollagen honeycomb scaffold

In order to induce new bone formation, mesenchymal stem cells were seeded onto atelocollagen honeycomb scaffold. We evaluated the mechanism of bone induction by KUSA/A1 cells combined with honeycomb atelocollagen scaffold. Scaffold alone, KUSA/A1 cells alone and with scaffold were implanted in the subcutaneous pockets of 4-week-old male SCID mice. The transplants were subjected to radiographical, histological and immunohistochemical examinations after 2 and 4 weeks of implantation. Radiographically, both KUSA/A1 cells alone and KUSA/A1-Scaffold showed some radiopaque areas formation but the latter disclosed a larger amount. Scaffold alone did not show any radiopacity. Histologically, Scaffold alone demonstrated only fibrous connective tissues in the periphery of the scaffold. KUSA/A1 cells alone showed few small islands of new bone formation surrounded by a thin layer of cellular proliferation. On the other hand, KUSA/A1-Scaffold revealed abundant new bone formation as well as cellular proliferation. We also determined the immunolocalization of type I collagen, CD34, Osteocalcin and PCNA in this newly formed bone. Our results indicated that less amount of stem cells are capable to induce the more amount of new bone in tissue engineering. This study support that atelocollagen honeycomb scaffold plays an important role in cellular anchorage and in vessel invasion, giving the precise shape and size for the new bone formation.     

Concepts of scaffold-based tissue engineering--the rationale to use solid free-form fabrication techniques

A paradigm shift is taking place in orthopaedic and reconstructive surgery from using medical devices and tissue grafts to a tissue engineering approach that uses biodegradable scaffolds combined with cells or biological molecules to repair and/or regenerate tissues. One of the potential benefits offered by solid free-form fabrication technology (SFF) is the ability to create scaffolds with highly reproducible architecture and compositional variation across the entire scaffold, due to its tightly controlled computer-driven fabrication. In this review, we define scaffold properties and attempt to provide some broad criteria and constraints for scaffold design in bone engineering.We also discuss the application-specific modifications driven by surgeon's requirements in vitro and/or in vivo. Next, we review the current use of SFF techniques in scaffold fabrication in the context of their clinical use in bone regeneration. Lastly, we comment on future developments in our groups, such as the functionalization of novel composite scaffolds with combinations of growth factors; and more specifically the promising area of heparan sulphate polysaccharide immobilization within the bone tissue engineering arena.