Taking cues from the extracellular matrix to design bone-mimetic regenerative scaffolds

There is an ongoing need for effective materials that can replace autologous bone grafts in the clinical treatment of bone injuries and deficiencies. In recent years, research efforts have shifted away from a focus on inert biomaterials to favor scaffolds that mimic the biochemistry and structure of the native bone extracellular matrix (ECM). The expectation is that such scaffolds will integrate with host tissue and actively promote osseous healing. To further enhance the osteoinductivity of bone graft substitutes, ECM-mimetic scaffolds are being engineered with a range of growth factors (GFs). The technologies used to generate GF-modified scaffolds are often inspired by natural processes that regulate the association between endogenous ECMs and GFs. The purpose of this review is to summarize research centered on the development of regenerative scaffolds that replicate the fundamental collagen-hydroxyapatite structure of native bone ECM, and the functionalization of these scaffolds with GFs that stimulate critical events in osteogenesis.     

SEM and 3D synchrotron radiation micro-tomography in the study of bioceramic scaffolds for tissue-engineering applications

Different biomaterials have been proposed as scaffolds for the delivery of cells and/or biological molecules to repair or regenerate damaged or diseased bone tissues. Particular attention is being given to porous bioceramics that mimic trabecular bone chemistry and structure. Chemical composition, density, pore shape, pore size, and pore interconnection are elements that have to be considered to improve the efficiency of these biomaterials. Commonly, two-dimensional (2D) systems of analysis such as scanning electron microscope (SEM) are used for the characterization and comparison of the scaffolds. Unfortunately, these systems do not allow a complete investigation of the three-dimensional (3D) spatial structure of the scaffold. In this study, we have considered two different techniques, that is, SEM and 3D synchrotron radiation (SR) micro-CT to extract information on the geometry of two hydroxyapatite (HA) bioceramics with identical chemical composition but different micro-porosity, pore size distribution, and pore interconnection pathway. The two scaffolds were obtained with two different procedures: (a) sponge matrix embedding (scaffold FB), and (b) foaming (scaffold EP). Both scaffolds showed structures suitable for tissue-engineering applications, but scaffold EP appeared superior with regard to interconnection of pores, surface on which the new bone could be deposited, and percentage of volume available to bone deposition.     

Novel peptide-based biomaterial scaffolds for tissue engineering.

Biomaterial scaffolds are components of cell-laden artificial tissues and transplantable biosensors. Some of the most promising new synthetic biomaterial scaffolds are composed of self-assembling peptides that can be modified to contain biologically active motifs. Peptide-based biomaterials can be fabricated to form two- and three-dimensional structures. Recent studies show that biomaterial promotion of multi-dimensional cell-cell interactions and cell density are crucial for proper cellular differentiation and for subsequent tissue formation. Other refinements in tissue engineering include the use of stem cells, cell pre-selection and growth factor pre-treatment of cells that are used for seeding scaffolds. These cell-culture technologies, combined with improved processes for defining the dimensions of peptide-based scaffolds, might lead to further improvements in tissue engineering. Novel peptide-based biomaterial scaffolds seeded with cells show promise for tissue repair and for other medical applications.     

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.     

Determining a Clinically Relevant Strategy for Bone Tissue Engineering: An “All-in-One” Study in Nude Mice

Purpose

Autologous bone grafting (BG) remains the standard reconstruction strategy for large craniofacial defects. Calcium phosphate (CaP) biomaterials, such as biphasic calcium phosphate (BCP), do not yield consistent results when used alone and must then be combined with cells through bone tissue engineering (BTE). In this context, total bone marrow (TBM) and bone marrow-derived mesenchymal stem cells (MSC) are the primary sources of cellular material used with biomaterials. However, several other BTE strategies exist, including the use of growth factors, various scaffolds, and MSC isolated from different tissues. Thus, clinicians might be unsure as to which method offers patients the most benefit. For this reason, the aim of this study was to compare eight clinically relevant BTE methods in an “all-in-one” study.

Methods

We used a transgenic rat strain expressing green fluorescent protein (GFP), from which BG, TBM, and MSC were harvested. Progenitor cells were then mixed with CaP materials and implanted subcutaneously into nude mice. After eight weeks, bone formation was evaluated by histology and scanning electron microscopy, and GFP-expressing cells were tracked with photon fluorescence microscopy.

Results/Conclusions

Bone formation was observed in only four groups. These included CaP materials mixed with BG or TBM, in which abundant de novo bone was formed, and BCP mixed with committed cells grown in two- and three-dimensions, which yielded limited bone formation. Fluorescence microscopy revealed that only the TBM and BG groups were positive for GFP expressing-cells, suggesting that these donor cells were still present in the host and contributed to the formation of bone. Since the TBM-based procedure does not require bone harvest or cell culture techniques, but provides abundant de novo bone formation, we recommend consideration of this strategy for clinical applications.     

Bio-composites composed of a solid free-form fabricated polycaprolactone and alginate-releasing bone morphogenic protein and bone formation peptide for bone tissue regeneration

Biomedical scaffolds should be designed with highly porous three-dimensional (3D) structures that have mechanical properties similar to the replaced tissue, biocompatible properties, and biodegradability. Here, we propose a new composite composed of solid free-form fabricated polycaprolactone (PCL), bone morphogenic protein (BMP-2) or bone formation peptide (BFP-1), and alginate for bone tissue regeneration. In this study, PCL was used as a mechanical supporting component to enhance the mechanical properties of the final biocomposite and alginate was used as the deterring material to control the release of BMP-2 and BFP-1. A release test revealed that alginate can act as a good release control material. The in vitro biocompatibilities of the composites were examined using osteoblast-like cells (MG63) and the alkaline phosphatase (ALP) activity and calcium deposition were assessed. The in vitro test results revealed that PCL/BFP-1/Alginate had significantly higher ALP activity and calcium deposition than the PCL/BMP-2/Alginate composite. Based on these findings, release-controlled BFP-1 could be a good growth factor for enhancement of bone tissue growth and the simple-alginate coating method will be a useful tool for fabrication of highly functional biomaterials through release–control supplementation.     

Use of high-resolution MRI for investigation of fluid flow and global permeability in a material with interconnected porosity

We present a multi-scale experimental approach designed to improve the investigation of both localized and global fluid flow in biomaterials with randomly interconnected porosity. Coralline hydroxyapatite (ProOsteon 500 from Interpore-Cross), having a relatively well-defined porosity, was used as an in vitro model of typical bone architecture. Axial fluid velocity profiles within the pores of a cylindrical hydroxyapatite sample were characterized using high-resolution MRI in conjunction with the measurement of global flow and associated permeability based on the Darcy-type relationship. Assuming Newtonian fluid behaviour, image analysis permitted computation of local porosity, intra-pore fluid shear, and visualization of flow heterogeneity within the sample. These results may benefit applications in biomaterials for the evaluation of factors influencing bony incorporation in porous scaffolds and on porous implant and bone surfaces. Normal and diseased biological tissues are also clinical relevant applications.     

Differentiation of bone marrow-derived mesenchymal stem cells into hepatocyte-like cells in an alginate scaffold

Objectives: Alginate scaffolds are the most frequently investigated biomaterials in tissue engineering. Tissue engineering techniques that generate liver tissue have become important for treatment of a number of liver diseases and recent studies indicate that bone marrow‐derived stem cells (BMSCs) can differentiate into hepatocyte‐like cells. The goal of the study described here, was to examine in vitro hepatic differentiation potential of BMSCs cultured in an alginate scaffold. Materials and methods: To investigate the potential of BMSCs to differentiate into hepatocyte‐like cells, we cultured BMSCs in alginate scaffolds in the presence of specific growth factors including hepatocyte growth factor, epidermal growth factor and fibroblast growth factor‐4. Results: We can demonstrate that alginate scaffolds are compatible for growth of BMSCs and when cultured in alginate scaffolds for several days they display several liver‐specific markers and functions. Specifically, they expressed genes encoding alpha‐foetoprotein, albumin (ALB), connexin 32 and CYP7A1. In addition, these BMSCs produced both ALB and urea, expressed cytokeratin‐18 (CK‐18) and were capable of glycogen storage. Percentage of CK‐18 positive cells, a marker of hepatocytes, was 56.7%. Conclusions: Our three‐dimensional alginate scaffolds were highly biocompatible with BMSCs. Furthermore, culturing induced their differentiation into hepatocyte‐like cells. Therefore, BMSCs cultured in alginate scaffolds may be applicable for hepatic tissue engineering.     

Segmental bone defects: from cellular and molecular pathways to the development of novel biological treatments

Several conditions in clinical orthopaedic practice can lead to the development of a diaphyseal segmental bone defect, which cannot heal without intervention. Segmental bone defects have been traditionally treated with bone grafting and/or distraction osteogenesis, methods that have many advantages, but also major drawbacks, such as limited availability, risk of disease transmission and prolonged treatment. In order to overcome such limitations, biological treatments have been developed based on specific pathways of bone physiology and healing. Bone tissue engineering is a dynamic field of research, combining osteogenic cells, osteoinductive factors, such as bone morphogenetic proteins, and scaffolds with osteoconductive and osteoinductive attributes, to produce constructs that could be used as bone graft substitutes for the treatment of segmental bone defects. Scaffolds are usually made of ceramic or polymeric biomaterials, or combinations of both in composite materials. The purpose of the present review is to discuss in detail the molecular and cellular basis for the development of bone tissue engineering constructs.     

Advances in 3D printing of composite scaffolds for the repairment of bone tissue associated defects

The conventional methods of using autografts and allografts for repairing defects in bone, the osteochondral bone, and the cartilage tissue have many disadvantages, like donor site morbidity and shortage of donors. Moreover, only 30% of the implanted grafts are shown to be successful in treating the defects. Hence, exploring alternative techniques such as tissue engineering to treat bone tissue associated defects is promising as it eliminates the above-mentioned limitations. To enhance the mechanical and biological properties of the tissue engineered product, it is essential to fabricate the scaffold used in tissue engineering by the combination of various biomaterials. Three-dimensional (3D) printing, with its ability to print composite materials and with complex geometry seems to have a huge potential in scaffold fabrication technique for engineering bone associated tissues. This review summarizes the recent applications and future perspectives of 3D printing technologies in the fabrication of composite scaffolds used in bone, osteochondral, and cartilage tissue engineering. Key developments in the field of 3D printing technologies involves the incorporation of various biomaterials and cells in printing composite scaffolds mimicking physiologically relevant complex geometry and gradient porosity. Much recently, the emerging trend of printing smart scaffolds which can respond to external stimulus such as temperature, pH and magnetic field, known as 4D printing is gaining immense popularity and can be considered as the future of 3D printing applications in the field of tissue engineering.     

Inductive tissue engineering with protein and DNA-releasing scaffolds

Cellular differentiation, organization, proliferation and apoptosis are determined by a combination of an intrinsic genetic program, matrix/substrate interactions, and extracellular cues received from the local microenvironment. These molecular cues come in the form of soluble (e.g. cytokines) and insoluble (e.g. ECM proteins) factors, as well as signals from surrounding cells that can promote specific cellular processes leading to tissue formation or regeneration. Recent developments in the field of tissue engineering have employed biomaterials to present these cues, providing powerful tools to investigate the cellular processes involved in tissue development, or to devise therapeutic strategies based on cell replacement or tissue regeneration. These inductive scaffolds utilize natural and/or synthetic biomaterials fabricated into three-dimensional structures. This review summarizes the use of scaffolds in the dual role of structural support for cell growth and vehicle for controlled release of tissue inductive factors, or DNA encoding for these factors. The confluence of molecular and cell biology, materials science and engineering provides the tools to create controllable microenvironments that mimic natural developmental processes and direct tissue formation for experimental and therapeutic applications.     

Human Urine Derived Stem Cells in Combination with β-TCP Can Be Applied for Bone Regeneration

Bone tissue engineering requires highly proliferative stem cells that are easy to isolate. Human urine stem cells (USCs) are abundant and can be easily harvested without using an invasive procedure. In addition, in our previous studies, USCs have been proved to be able to differentiate into osteoblasts, chondrocytes, and adipocytes. Therefore, USCs may have great potential and advantages to be applied as a cell source for tissue engineering. However, there are no published studies that describe the interactions between USCs and biomaterials and applications of USCs for bone tissue engineering. Therefore, the objective of the present study was to evaluate the interactions between USCs with a typical bone tissue engineering scaffold, beta-Tricalcium Phosphate (β-TCP), and to determine whether the USCs seeded onto β-TCP scaffold can promote bone regeneration in a segmental femoral defect of rats. Primary USCs were isolated from urine and seeded on β-TCP scaffolds. Results showed that USCs remained viable and proliferated within β-TCP. The osteogenic differentiation of USCs within the scaffolds was demonstrated by increased alkaline phosphatase activity and calcium content. Furthermore, β-TCP with adherent USCs (USCs/β-TCP) were implanted in a 6-mm critical size femoral defect of rats for 12 weeks. Bone regeneration was determined using X-ray, micro-CT, and histologic analyses. Results further demonstrated that USCs in the scaffolds could enhance new bone formation, which spanned bone defects in 5 out of 11 rats while β-TCP scaffold alone induced modest bone formation. The current study indicated that the USCs can be used as a cell source for bone tissue engineering as they are compatible with bone tissue engineering scaffolds and can stimulate the regeneration of bone in a critical size bone defect.     

Improvement of human skin cell growth by radiation-induced modifications of a Ge/Ch/Ha scaffold

Gelatin-/chitosan-/hyaluronan-based biomaterials are used in tissue engineering as cell scaffolds. Three gamma radiation doses (1, 10 and 25 kGy) were applied to scaffolds for sterilization. Microstructural changes of the irradiated polymers were evaluated by using scanning electron microscopy (SEM) and differential scanning calorimetry (DSC). A dose of 25 kGy produced a rough microstructure with a reduction of the porosity (from 99 to 96 %) and pore size (from 160 to 123 μm). Radiation also modified the glass transition temperature between 31.2 and 42.1 °C (1 and 25 kGy respectively). Human skin cells cultivated on scaffolds irradiated with 10 and 25 kGy proliferated at 48 h and secreted transforming growth factor β3 (TGF-β3). Doses of 0 kGy (non-irradiated) or 1 kGy did not stimulate TGF-β3 secretion or cell proliferation. The specific growth rate and lactate production increased proportionally to radiation dose. The use of an appropriate radiation dose improves the cell scaffold properties of biomaterials.     

Laser Photoablation of Guidance Microchannels into Hydrogels Directs Cell Growth in Three Dimensions

Recent years have seen rapid progress in the engineering and application of biomaterials with controlled biological, physical, and chemical properties, and the development of associated methods for micropatterning of three-dimensional tissue-engineering scaffolds. A remaining challenge is the development of robust, flexible methods that can be used to create physical guidance structures in cell-seeded scaffolds independently of environmental constraints. Here we demonstrate that focal photoablation caused by pulsed lasers can generate guidance structures in transparent hydrogels, with feature control down to the micron scale. These photopatterned microchannels guide the directional growth of neurites from dorsal root ganglia. We characterize the effect of laser properties and biomaterial properties on microchannel formation in PEGylated fibrinogen hydrogels, and the effect of photoablation on neural outgrowth. This strategy could lead to the development of a new generation of guidance channels for treating nerve injuries, and the engineering of structured three-dimensional neuronal or nonneuronal networks.     

Preparation of 3D fibrin scaffolds for stem cell culture applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue. A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside. This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach.     

Cell culture in autologous fibrin scaffolds for applications in tissue engineering

In tissue engineering techniques, three-dimensional scaffolds are needed to adjust and guide cell growth and to allow tissue regeneration. The scaffold must be biocompatible, biodegradable and must benefit the interactions between cells and biomaterial. Some natural biomaterials such as fibrin provide a structure similar to the native extracellular matrix containing the cells. Fibrin was first used as a sealant based on pools of commercial fibrinogen. However, the high risk of viral transmission of these pools led to the development of techniques of viral inactivation and elimination and the use of autologous fibrins. In recent decades, fibrin has been used as a release system and three-dimensional scaffold for cell culture. Fibrin scaffolds have been widely used for the culture of different types of cells, and have found several applications in tissue engineering. The structure and development of scaffolds is a key point for cell culture because scaffolds of autologous fibrin offer an important alternative due to their low fibrinogen concentrations, which are more suitable for cell growth.     

Suspension-adapted Chinese hamster ovary-derived cells expressing green fluorescent protein as a screening tool for biomaterials

Synthetic biomaterials play an important role in regenerative medicine. To be effective they must support cell attachment and proliferation in addition to being non-toxic and non-immunogenic. We used a suspension-adapted Chinese hamster ovary-derived cell line expressing green fluorescent protein (GFP) to assess cell attachment and growth on synthetic biomaterials by direct measurement of GFP-specific fluorescence. To simplify operations, all cell cultivation steps were performed in orbitally-shaken, disposable containers. Comparative studies between this GFP assay and previously established cell quantification assays demonstrated that this novel approach is suitable for rapid screening of a large number of samples. Furthermore the utility of our assay system was confirmed by evaluation of cell growth on three polyvinylidene fluoride polymer scaffolds that differed in pore diameter and drawing conditions. The data presented here prove the general utility of GFP-expressing cell lines and orbital shaking technology for the screening of biomaterials for tissue engineering applications.     

WJSC 6th Anniversary Special Issues（2）:Mesenchymal stem cellsAdipose mesenchymal stem cells in the field of bone tissue engineering

Bone tissue engineering represents one of the most challenging emergent fields for scientists and clinicians.Current failures of autografts and allografts in many pathological conditions have prompted researchers to find new biomaterials able to promote bone repair or regeneration with specific characteristics of biocompatibility,biodegradability and osteoinductivity.Recent advancements for tissue regeneration in bone defects have occurred by following the diamond concept and combining the use of growth factors and mesenchymal stem cells(MSCs).In particular,a more abundant and easily accessible source of MSCs was recently discovered in adipose tissue.These adipose stem cells(ASCs)can be obtained in large quantities with little donor site morbidity or patient discomfort,in contrast to the invasive and painful isolation of bone marrow MSCs.The osteogenic potential of ASCs on scaffolds has been examined in cell cultures and animal models,with only a few cases reporting the use of ASCs for successful reconstruction or accelerated healing of defects of the skull and jaw in patients.Although these reports extend our limited knowledge concerning the use of ASCs for osseous tissue repair and regeneration,the lack of standardization in applied techniques makes the comparison between studies difficult.Additional clinical trials are needed to assess ASC therapy and address potential ethical and safety concerns,which must be resolved to permit application in regenerative medicine.     

Biodegradable bone regeneration synthetic scaffolds: in tissue engineering

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

Directed assembly of cell-laden hydrogels for engineering functional tissues

Tissue engineering aims to develop functionalized tissues for organ replacement or restoration. Biodegradable scaffolds have been used in tissue engineering to support cell growth and maintain mechanical and biological properties of tissue constructs. Ideally cells on these scaffolds adhere, proliferate, and deposit matrix at a rate that is consistent with scaffold degradation. However, the cellular rearrangement within these scaffolds often does not recapitulate the architecture of the native tissues. Directed assembly of tissue-like structures is an attractive alternative to scaffold-based approach for tissue engineering which potentially can build tissue constructs with biomimetic architecture and function. In directed assembly, shape-controlled microstructures are fabricated in which organized structures of different cell types can be used as tissue building blocks. To fabricate tissue building blocks, hydrogels are commonly used as biomaterials for cell encapsulation to mimic the matrix in vivo. The hydrogel-based tissue building blocks can be arranged in pre-defined architectures by various directed tissue assembly techniques. In this paper, recent advances in directed assembly-based tissue engineering are summarized as an emerging alternative to meet challenges associated with scaffold-based tissue engineering and future directions are addressed.