Mesoporous bioglass/silk composite scaffolds for bone tissue engineering

In the past 20 years, mesoporous materials have been attracted great attention due to their significant feature of large surface area, ordered mesoporous structure, tunable pore size and volume, and well-defined surface property. They have many potential applications, such as catalysis, adsorption/separation, biomedicine, etc. [1]. Recently, the studies of the applications of mesoporous materials have been expanded into the field of biomaterials science. A new class of bioactive glass, referred to as mesoporous bioactive glass (MBG), was first developed in 2004. This material has a highly ordered mesopore channel structure with a pore size ranging from 5–20 nm [1]. Compared to non-mesopore bioactive glass (BG), MBG possesses a more optimal surface area, pore volume and improved in vitro apatite mineralization in simulated body fluids [1,2]. Vallet-Regí et al. has systematically investigated the in vitro apatite formation of different types of mesoporous materials, and they demonstrated that an apatite-like layer can be formed on the surfaces of Mobil Composition of Matters (MCM)-48, hexagonal mesoporous silica (SBA-15), phosphorous-doped MCM-41, bioglass-containing MCM-41 and ordered mesoporous MBG, allowing their use in biomedical engineering for tissue regeneration [2-4]. Chang et al. has found that MBG particles can be used for a bioactive drug-delivery system [5,6]. Our study has shown that MBG powders, when incorporated into a poly (lactide-co-glycolide) (PLGA) film, significantly enhance the apatite-mineralization ability and cell response of PLGA films. compared to BG [7]. These studies suggest that MBG is a very promising bioactive material with respect to bone regeneration. It is known that for bone defect repair, tissue engineering represents an optional method by creating three-dimensional (3D) porous scaffolds which will have more advantages than powders or granules as 3D scaffolds will provide an interconnected macroporous network to allow cell migration, nutrient delivery, bone ingrowth, and eventually vascularization [8]. For this reason, we try to apply MBG for bone tissue engineering by developing MBG scaffolds. However, one of the main disadvantages of MBG scaffolds is their low mechanical strength and high brittleness; the other issue is that they have very quick degradation, which leads to an unstable surface for bone cell growth limiting their applications. Silk fibroin, as a new family of native biomaterials, has been widely studied for bone and cartilage repair applications in the form of pure silk or its composite scaffolds [9-14]. Compared to traditional synthetic polymer materials, such as PLGA and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), the chief advantage of silk fibroin is its water-soluble nature, which eliminates the need for organic solvents, that tend to be highly cytotoxic in the process of scaffold preparation [15]. Other advantages of silk scaffolds are their excellent mechanical properties, controllable biodegradability and cytocompatibility [15-17]. However, for the purposes of bone tissue engineering, the osteoconductivity of pure silk scaffolds is suboptimal. It is expected that combining MBG with silk to produce MBG/silk composite scaffolds would greatly improve their physiochemical and osteogenic properties for bone tissue engineering application. Therefore, in this chapter, we will introduce the research development of MBG/silk scaffolds for bone tissue engineering.

Mesenchymal stem cells in osteoarthritis therapy : current status of theory, technology, and applications

Osteoarthritis (OA) is a chronic, non-inflammatory type of arthritis, which usually affects the movable and weight bearing joints of the body. It is the most common joint disease in human beings and common in elderly people. Till date, there are no safe and effective diseases modifying OA drugs (DMOADs) to treat the millions of patients suffering from this serious and debilitating disease. However, recent studies provide strong evidence for the use of mesenchymal stem cell (MSC) therapy in curing cartilage related disorders. Due to their natural differentiation properties, MSCs can serve as vehicles for the delivery of effective, targeted treatment to damaged cartilage in OA disease. In vitro, MSCs can readily be tailored with transgenes with anti-catabolic or pro-anabolic effects to create cartilage-friendly therapeutic vehicles. On the other hand, tissue engineering constructs with scaffolds and biomaterials holds promising biological cartilage therapy. Many of these strategies have been validated in a wide range of in vitro and in vivo studies assessing treatment feasibility or efficacy. In this review, we provide an outline of the rationale and status of stem-cell-based treatments for OA cartilage, and we discuss prospects for clinical implementation and the factors crucial for maintaining the drive towards this goal.

Instability prolongs the chondral phase during bone healing in sheep

In this sheep study, we investigated the influence of fixation stability on the temporal and spatial distribution of tissues in the fracture callus. As the initial mechanical conditions have been cited as being especially important for the healing outcome, it was hypothesized that differences in the path of healing would be seen as early as the initial phase of healing. ----- ----- Sixty-four sheep underwent a mid-shaft tibial osteotomy that was treated with either a rigid or a semi-rigid external fixator. Animals were sacrificed at 2, 3, 6 and 9 weeks postoperatively and the fracture calluses were analyzed using radiological, biomechanical and histological techniques. Statistical comparison between the groups was performed using the Mann–Whitney U test for unpaired non-parametric data. ----- ----- In the callus of the tibia treated with semi-rigid fixation, remnants of the fracture haematoma remained present for longer, although new periosteal bone formation during early healing was similar in both groups. The mechanical competence of the healing callus at 6 weeks was inferior compared to tibiae treated with rigid fixation. Semi-rigid fixation resulted in a larger cartilage component of the callus, which persisted longer. Remodeling processes were initiated earlier in the rigid group, while new bone formation continued throughout the entire investigated period in the semi-rigid group. ----- ----- In this study, evidence is provided that less rigid fixation increased the time required for healing. The process of intramembranous ossification appeared during the initial stages of healing to be independent of mechanical stability. However, the delay in healing was related to a prolonged chondral phase.

The mechanical heterogeneity of the hard callus influences local tissue strains during bone healing : a finite element study based on sheep experiments

During secondary fracture healing, various tissue types including new bone are formed. The local mechanical strains play an important role in tissue proliferation and differentiation. To further our mechanobiological understanding of fracture healing, a precise assessment of local strains is mandatory. Until now, static analyses using Finite Elements (FE) have assumed homogenous material properties. With the recent quantification of both the spatial tissue patterns (Vetter et al., 2010) and the development of elastic modulus of newly formed bone during healing (Manjubala et al., 2009), it is now possible to incorporate this heterogeneity. Therefore, the aim of this study is to investigate the effect of this heterogeneity on the strain patterns at six successive healing stages. The input data of the present work stemmed from a comprehensive cross-sectional study of sheep with a tibial osteotomy (Epari et al., 2006). In our FE model, each element containing bone was described by a bulk elastic modulus, which depended on both the local area fraction and the local elastic modulus of the bone material. The obtained strains were compared with the results of hypothetical FE models assuming homogeneous material properties. The differences in the spatial distributions of the strains between the heterogeneous and homogeneous FE models were interpreted using a current mechanobiological theory (Isakson et al., 2006). This interpretation showed that considering the heterogeneity of the hard callus is most important at the intermediate stages of healing, when cartilage transforms to bone via endochondral ossification.

Temporal tissue patterns in bone healing of sheep

Secondary fracture healing in long bones leads to the successive formation of intricate patterns of tissues in the newly formed callus. The main aim of this work was to quantitatively describe the topology of these tissue patterns at different stages of the healing process and to generate averaged images of tissue distribution. This averaging procedure was based on stained histological sections (2, 3, 6, and 9 weeks post-operatively) of 64 sheep with a 3 mm tibial mid-shaft osteotomy, stabilized either with a rigid or a semi-rigid external fixator. Before averaging, histological images were sorted for topology according to six identified tissue patterns. The averaged images were obtained for both fixation types and the lateral and medial side separately. For each case, the result of the averaging procedure was a collection of six images characterizing quantitatively the progression of the healing process. In addition, quantified descriptions of the newly formed cartilage and the bone area fractions (BA/TA) of the bony callus are presented. For all cases, a linear increase in the BA/TA of the bony callus was observed. The slope was greatest in the case of the most rigid stabilization and lowest in the case of the least stiff. This topological description of the progression of bone healing will allow quantitative validation (or falsification) of current mechano-biological theories.

Endochondral ossification in vitro is influenced by mechanical bending

Bone development is influenced by the local mechanical environment. Experimental evidence suggests that altered loading can change cell proliferation and differentiation in chondro- and osteogenesis during endochondral ossification. This study investigated the effects of three-point bending of murine fetal metatarsal bone anlagen in vitro on cartilage differentiation, matrix mineralization and bone collar formation. This is of special interest because endochondral ossification is also an important process in bone healing and regeneration. Metatarsal preparations of 15 mouse fetuses stage 17.5 dpc were dissected en bloc and cultured for 7 days. After 3 days in culture to allow adherence they were stimulated 4 days for 20 min twice daily by a controlled bending of approximately 1000-1500 microstrain at 1 Hz. The paraffin-embedded bone sections were analyzed using histological and histomorphometrical techniques. The stimulated group showed an elongated periosteal bone collar while the total bone length was not different from controls. The region of interest (ROI), comprising the two hypertrophic zones and the intermediate calcifying diaphyseal zone, was greater in the stimulated group. The mineralized fraction of the ROI was smaller in the stimulated group, while the absolute amount of mineralized area was not different. These results demonstrate that a new device developed to apply three-point bending to a mouse metatarsal bone culture model caused an elongation of the periosteal bone collar, but did not lead to a modification in cartilage differentiation and matrix mineralization. The results corroborate the influence of biophysical stimulation during endochondral bone development in vitro. Further experiments with an altered loading regime may lead to more pronounced effects on the process of endochondral ossification and may provide further insights into the underlying mechanisms of mechanoregulation which also play a role in bone regeneration.

CYR61 (CCN1) protein expression during fracture healing in an ovine tibial model and its relation to the mechanical fixation stability

The formation of new blood vessels is a prerequisite for bone healing. CYR61 (CCN1), an extracellular matrix-associated signaling protein, is a potent stimulator of angiogenesis and mesenchymal stem cell expansion and differentiation. A recent study showed that CYR61 is expressed during fracture healing and suggested that CYR61 plays a significant role in cartilage and bone formation. The hypothesis of the present study was that decreased fixation stability, which leads to a delay in healing, would lead to reduced CYR61 protein expression in fracture callus. The aim of the study was to quantitatively analyze CYR61 protein expression, vascularization, and tissue differentiation in the osteotomy gap and relate to the mechanical fixation stability during the course of healing. A mid-shaft osteotomy of the tibia was performed in two groups of sheep and stabilized with either a rigid or semirigid external fixator, each allowing different amounts of interfragmentary movement. The sheep were sacrificed at 2, 3, 6, and 9 weeks postoperatively. The tibiae were tested biomechanically and histological sections from the callus were analyzed immunohistochemically with regard to CYR61 protein expression and vascularization. Expression of CYR61 protein was upregulated at the early phase of fracture healing (2 weeks), decreasing over the healing time. Decreased fixation stability was associated with a reduced upregulation of the CYR61 protein expression and a reduced vascularization at 2 weeks leading to a slower healing. The maximum cartilage callus fraction in both groups was reached at 3 weeks. However, the semirigid fixator group showed a significantly lower CYR61 immunoreactivity in cartilage than the rigid fixator group at this time point. The fraction of cartilage in the semirigid fixator group was not replaced by bone as quickly as in the rigid fixator group leading to an inferior histological and mechanical callus quality at 6 weeks and therefore to a slower healing. The results supply further evidence that CYR61 may serve as an important regulator of bone healing.

Altered cell interactions of subchondral bone osteoblasts and articular chondrocytes in osteoarthritis is through the mediation of ERK1/2 phosphorylation

Introduction: Osteoarthritis (OA) is the most common musculoskeletal disorder and represents a major health burden to society. In the course of the pathological development of OA, articular cartilage chondrocytes (ACCs) undergo a typical phenotype changes characterized by the expression of hypertrophic differentiation markers. Also, the adjacent subchondral bone shows signs of abnormal mineral density and enhanced production of bone turnover markers, indicative of osteoblast dysfunction. However, the mechanism(s) by which these changes occur during the OA development are not completely understood. Materials and Methods: ACCs and subchondral bone osteoblasts (SBOs) were harvested from OA and healthy patients for the cross-talk studies between normal and OA ACCs and SBOs. The involvement of mitogen activated protein kinase (MAPK) signalling pathway during the cell-cell interactions was analysed by zymography, ELISA and western blotting methods. Results: The direct and in-direct co-culture studies showed that OA (ACCs and SBOs) cells induced osteoarthritic changes of normal (ACC and SBOs) cells. This altered cell interaction induced by OA cells significantly aggravated the proteolytic activity, which resulted cartilage degeneration. The altered cell interaction appeared to significantly activate ERK 1/2 phosphorylation and inhibition of MAPK-ERK 1/2 pathway reversed the osteoarthrtitic phenotypic changes. Discussion and Conclusion: Our study has demonstrated that the altered bi-directional communication of SBOs and ACCs are critical for initiation and progression of OA related changes and that this process is mediated by MAPK signalling pathways. Targeting these altered interactions by the use of MAPK inhibitors may provide the scientific rationale for the development of novel therapeutic strategies in the treatment and management of OA related disorders.

Long-term effects of hydrogel properties on human chondrocyte behavior

Hydrogels provide a 3-dimensional network for embedded cells and offer promise for cartilage tissue engineering applications. Nature-derived hydrogels, including alginate, have been shown to enhance the chondrocyte phenotype but are variable and not entirely controllable. Synthetic hydrogels, including polyethylene glycol (PEG)-based matrices, have the advantage of repeatability and modularity; mechanical stiffness, cell adhesion, and degradability can be altered independently. In this study, we compared the long-term in vitro effects of different hydrogels (alginate and Factor XIIIa-cross-linked MMP-sensitive PEG at two stiffness levels) on the behavior of expanded human chondrocytes and the development of construct properties. Monolayer-expanded human chondrocytes remained viable throughout culture, but morphology varied greatly in different hydrogels. Chondrocytes were characteristically round in alginate but mostly spread in PEG gels at both concentrations. Chondrogenic gene (COL2A1, aggrecan) expression increased in all hydrogels, but alginate constructs had much higher expression levels of these genes (up to 90-fold for COL2A1), as well as proteoglycan 4, a functional marker of the superficial zone. Also, chondrocytes expressed COL1A1 and COL10A1, indicative of de-differentiation and hypertrophy. After 12 weeks, constructs with lower polymer content were stiffer than similar constructs with higher polymer content, with the highest compressive modulus measured in 2.5% PEG gels. Different materials and polymer concentrations have markedly different potency to affect chondrocyte behavior. While synthetic hydrogels offer many advantages over natural materials such as alginate, they must be further optimized to elicit desired chondrocyte responses for use as cartilage models and for development of functional tissue-engineered articular cartilage.

Immunohistochemical analysis of structural changes in collagen for the assessment of osteoarthritis

Collagen fibrillation within articular cartilage (AC) plays a key role in joint osteoarthritis (OA) progression and, therefore, studying collagen synthesis changes could be an indicator for use in the assessment of OA. Various staining techniques have been developed and used to determine the collagen network transformation under microscopy. However, because collagen and proteoglycan coexist and have the same index of refraction, conventional methods for specific visualization of collagen tissue is difficult. This study aimed to develop an advanced staining technique to distinguish collagen from proteoglycan and to determine its evolution in relation to OA progression using optical and laser scanning confocal microscopy (LSCM). A number of AC samples were obtained from sheep joints, including both healthy and abnormal joints with OA grades 1 to 3. The samples were stained using two different trichrome methods and immunohistochemistry (IHC) to stain both colourimetrically and with fluorescence. Using optical microscopy and LSCM, the present authors demonstrated that the IHC technique stains collagens only, allowing the collagen network to be separated and directly investigated. Fluorescently-stained IHC samples were also subjected to LSCM to obtain three-dimensional images of the collagen fibres. Changes in the collagen fibres were then correlated with the grade of OA in tissue. This study is the first to successfully utilize the IHC staining technique in conjunction with laser scanning confocal microscopy. This is a valuable tool for assessing changes to articular cartilage in OA.

Adhesion of perichondrial cells to a polylactic acid scaffold

The number of chondrogenic cells available locally is an, important factor in the repair process for cartilage defects. Previous studies demonstrated that the number of transplanted rabbit perichondrial cells (PC) remaining in a cartilage defect in vivo, after being carried into the site in a polylactic acid (PLA) scaffold, declined markedly within two days. This study examined the ability of in vitro culture of PC/PLA constructs to enhance subsequent biomechanical stability of the cells and the matrix content in an in vitro screening assay. PC/PLA constructs were analyzed after 1 h, 1 and 2 weeks of culture. The biomechanical adherence of PC to the PLA scaffold was tested by subjecting the PC/PLA constructs to a range of flow velocities (0.25-25 mm/s), spanning the range estimated to occur under conditions of construct insertion in vivo. The adhesion of PC to the PLA carrier was increased significantly by 1 and 2 weeks of incubation, with 25 mm/s flow causing a 57% detachment of cells after 1 h of seeding, but only 7% and 16% after I and 2 weeks of culture, respectively (p < 0.001). This adherence was associated with marked deposition of glycosaminoglycan and collagen. These findings suggest that pre-incubation of PC-laden PLA scaffolds markedly enhances the stability of the indwelling cells. (C) 2003 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.

Cell density alters matrix accumulation in two distinct fractions and the mechanical integrity of alginate-chondrocyte constructs

Chondrocyte density in articular cartilage is known to change with the development and growth of the tissue and may play an important role in the formation of a functional extracellular matrix (ECM). The objective of this study was to determine how initial chondrocyte density in an alginate hydrogel affects the matrix composition, its distribution between the cell-associated (CM) and further removed matrix (FRM) fractions, and the tensile mechanical properties of the developing engineered cartilage. Alginate constructs containing primary bovine chondrocytes at densities of 0, 4, 16, and 64 million cells/ml were fabricated and cultured for 1 or 2 weeks, at which time structural, biochemical, and mechanical properties were analyzed. Both matrix content and distribution varied with the initial cell density. Increasing cell density resulted in an increasing content of collagen and sulfated-glycosaminoglycan (GAG) and an increasing proportion of these molecules localized in the CM. While the equilibrium tensile modulus of cell-free alginate did not change with time in culture, the constructs with highest cell density were 116% stiffer than cell-free controls after 2 weeks of culture. The equilibrium tensile modulus was positively correlated with total collagen (r2 = 0.47, p < 0.001) and GAG content (r2 = 0.68, p < 0.001), and these relationships were enhanced when analyzing only those matrix molecules in the CM fraction (r2 = 0.60 and 0.72 for collagen and GAG, respectively, each p < 0.001). Overall, the results of this study indicate that initial cell density has a considerable effect on the developing composition, structure, and function of alginate–chondrocyte constructs.

Short-term retention of labeled chondrocyte subpopulations in stratified tissue-engineered cartilaginous constructs implanted in vivo in mini-pigs

It is likely that effective application of cell-laden implants for cartilage defects depends on retention of implanted cells and interaction between implanted and host cells. The objectives of this study were to characterize stratified cartilaginous constructs seeded sequentially with superficial (S) and middle (M) chondrocyte subpopulations labelled with fluorescent cell tracking dye PKH26 (*) and determine the degree to which these stratified cartilaginous constructs maintain their architecture in vivo after implantation in mini-pigs for 1 week. Alginate-recovered cells were seeded sequentially to form stratified S*/M (only S cells labelled) and S*/M* (both S and M cells labelled) constructs. Full-thickness defects (4 mm diameter) were created in the patellofemoral groove of adult Yucatan mini-pigs and filled with portions of constructs or left empty. Constructs were characterized biochemically, histologically, and biomechanically, and stratification visualized and quantified, before and after implant. After 1 week, animals were sacrificed and implants retrieved. After 1 week in vivo, glycosaminoglycan and collagen content of constructs remained similar to that at implant, whereas DNA content increased. Histological analyses revealed features of an early repair response, with defects filled with tissues containing little matrix and abundant cells. Some implanted (PKH26-labeled) cells persisted in the defects, although constructs did not maintain a stratified organization. Of the labelled cells, 126 +/- 38% and 32 +/- 8% in S*/M and S*/M* constructs, respectively, were recovered. Distribution of labelled cells indicated interactions between implanted and host cells. Longer-term in vivo studies will be useful in determining whether implanted cells are sufficient to have a positive effect in repair.

Continuous passive motion applied to whole joints stimulates chondrocyte blosynthesis of PRG4

Continuous passive motion (CPM) is currently a part of patient rehabilitation regimens after a variety of orthopedic surgical procedures. While CPM can enhance the joint healing process, the direct effects of CPM on cartilage metabolism remain unknown. Recent in vivo and in vitro observations suggest that mechanical stimuli can regulate articular cartilage metabolism of proteoglycan 4 (PRG4), a putative lubricating and chondroprotective molecule found in synovial fluid and at the articular cartilage surface. ----- ----- Objectives: (1) Determine the topographical variation in intrinsic cartilage PRG4 secretion. (2) Apply a CPM device to whole joints in bioreactors and assess effects of CPM on PRG4 biosynthesis.----- ----- Methods: A bioreactor was developed to apply CPM to bovine stifle joints in vitro. Effects of 24 h of CPM on PRG4 biosynthesis were determined.----- ----- Results: PRG4 secretion rate varied markedly over the joint surface. Rehabilitative joint motion applied in the form of CPM regulated PRG4 biosynthesis, in a manner dependent on the duty cycle of cartilage sliding against opposing tissues. Specifically, in certain regions of the femoral condyle that were continuously or intermittently sliding against meniscus and tibial cartilage during CPM, chondrocyte PRG4 synthesis was higher with CPM than without.----- ----- Conclusions: Rehabilitative joint motion, applied in the form of CPM, stimulates chondrocyte PRG4 metabolism. The stimulation of PRG4 synthesis is one mechanism by which CPM may benefit cartilage and joint health in post-operative rehabilitation. (C) 2006 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

Zonal chondrocyte subpopulations reacquire zone-specific characteristics during in Vitro redifferentiation

Background: If chondrocytes from the superficial, middle, and deep zones of articular cartilage could maintain or regain their characteristic properties during in vitro culture, it would be feasible to create constructs comprising these distinctive zones. ----- ----- Hypothesis: Zone-specific characteristics of zonal cell populations will disappear during 2-dimensional expansion but will reappear after 3-dimensional redifferentiation, independent of the culture technique used (alginate beads versus pellet culture).----- ----- Study Design: Controlled laboratory study.----- ----- Methods: Equine articular chondrocytes from the 3 zones were expanded in monolayer culture (8 donors) and subsequently redifferentiated in pellet and alginate bead cultures for up to 4 weeks. Glycosaminoglycans and DNA were quantified, along with immunohistochemical assessment of the expression of various zonal markers, including cartilage oligomeric protein (marking cells from the deeper zones) and clusterin (specifically expressed by superficial chondrocytes).----- ----- Results: Cell yield varied between zones, but proliferation rates did not show significant differences. Expression of all evaluated zonal markers was lost during expansion. Compared to the alginate bead cultures, pellet cultures showed a higher amount of glycosaminoglycans produced per DNA after redifferentiation. In contrast to cells in pellet cultures, cells in alginate beads regained zonal differences, as evidenced by zone-specific reappearance of cartilage oligomeric protein and clusterin, as well as significantly higher glycosaminoglycans production by cells from the deep zone compared to the superficial zone.----- ----- Conclusion: Chondrocytes isolated from the 3 zones of equine cartilage can restore their zone-specific matrix expression when cultured in alginate after in vitro expansion.