Nano- to macroscale remodeling of functional tissue-engineered bone

A higher degree of mineralization is found within scaffold groups implanted with cells compared to scaffold alone demonstrating greater bone regenerative potential of cell-scaffold constructs Tissue engineered bone analysed using ESEM and SAXS demonstrates bone formation within the scaffold to be preferentially aligned around the scaffold struts. The mineral particles are not shown to orientate around the osteons within the native bone.

Comparative study of depth-dependent characteristics of equine and human osteochondral tissue from the medial and lateral femoral condyles

Articular cartilage defects are common after joint injuries. When left untreated, the biomechanical protective function of cartilage is gradually lost, making the joint more susceptible to further damage, causing progressive loss of joint function and eventually osteoarthritis (OA). In the process of translating promising tissue-engineering cartilage repair approaches from bench to bedside, pre-clinical animal models including mice, rabbits, goats, and horses, are widely used. The equine species is becoming an increasingly popular model for the in vivo evaluation of regenerative orthopaedic approaches. As there is also an increasing body of evidence suggesting that successful lasting tissue reconstruction requires an implant that mimics natural tissue organization, it is imperative that depth-dependent characteristics of equine osteochondral tissue are known, to assess to what extent they resemble those in humans. Therefore, osteochondral cores (4-8 mm) were obtained from the medial and lateral femoral condyles of equine and human donors. Cores were processed for histology and for biochemical quantification of DNA, glycosaminoglycan (GAG) and collagen content. Equine and human osteochondral tissues possess similar geometrical (thickness) and organizational (GAG, collagen and DNA distribution with depth) features. These comparable trends further underscore the validity of the equine model for the evaluation of regenerative approaches for articular cartilage.

In vitro physical stimulation of tissue-engineered and native cartilage

Because of the limited availability of donor cartilage for resurfacing defects in articular surfaces, there is tremendous interest in the in vitro bioengineering of cartilage replacements for clinical applications. However, attaining mechanical properties in engineered cartilaginous constructs that approach those of native cartilage has not been previously achieved when constructs are cultured under free-swelling conditions. One approach toward stimulating the development of constructs that are mechanically more robust is to expose them to physical environments that are similar, in certain ways, to those encountered by native cartilage. This is a strategy motivated by observations in numerous short-term experiments that certain mechanical signals are potent stimulators of cartilage metabolism. On the other hand, excess mechanical loading can have a deleterious effect on cartilage. Culture conditions that include a physical stimulation component are made possible by the use of specialized bioreactors. This chapter addresses some of the issues involved in using bioreactors as integral components of cartilage tissue engineering and in studying the physical regulation of cartilage. We first consider the generation of cartilaginous constructs in vitro. Next we describe the rationale and design of bioreactors that can impart either mechanical deformation or fluid-induced mechanical signals.

The key regulatory roles of the PI3K/Akt signalling pathway in the functionalities of mesenchymal stem cells and applications in tissue regeneration

Mesenchymal stem cells (MSCs) are multi-potent cells that can differentiate into various cell types and have been used widely in tissue engineering application. In tissue engineering, a scaffold, MSCs and growth factors are used as essential components and their interactions have been regarded to be important for regeneration of tissues. A critical problem for MSCs in tissue engineering is their low survival ability and functionality. Most MSCs are going to be apoptotic after transplantation. Therefore, increasing MSC survival ability and functionalities is the key for potential applications of MSCs. Several approaches have been studied to increase MSC tissue forming capacity including application of growth factors, overexpression of stem cell regulatory genes and improvement of biomaterials for scaffolds. The effects of these approaches on MSCs have been associated with the activation of the PI3K/Akt signaling pathway. The pathway plays central regulatory roles in MSC survival, proliferation, migration, angiogenesis, cytokine production and differentiation. In this review, we summarize and discuss the literatures related to the roles of the PI3K/Akt pathway in the functionalities of MSCs and the involvement of the pathway in biomaterials-increased MSC functinalities. Biomaterials have been modified in their properties, surface structure and loaded with growth factors to increase MSC functionalities. Several studies demonstrated that the biomaterials-increased MSC functionalities are mediated by the activation of the PI3K/Akt pathway.

Gelatin-methacrylamide hydrogels as potential biomaterials for fabrication of tissue-engineered cartilage constructs

Gelatin-methacrylamide (gelMA) hydrogels are shown to support chondrocyte viability and differentiation and give wide ranging mechanical properties depending on several cross-linking parameters. Polymer concentration, UV exposure time, and thermal gelation prior to UV exposure allow for control over hydrogel stiffness and swelling properties. GelMA solutions have a low viscosity at 37 °C, which is incompatible with most biofabrication approaches. However, incorporation of hyaluronic acid (HA) and/or co-deposition with thermoplastics allows gelMA to be used in biofabrication processes. These attributes may allow engineered constructs to match the natural functional variations in cartilage mechanical and geometrical properties.

Cross-linked poly(trimethylene carbonate-co-L-lactide) as a biodegradable, elastomeric scaffold for vascular engineering applications

A series of copolymers of trimethylene carbonate (TMC) and l-lactide (LLA) were synthesized and evaluated as scaffolds for the production of artificial blood vessels. The polymers were end-functionalized with acrylate, cast into films, and cross-linked using UV light. The mechanical, degradation, and biocompatibility properties were evaluated. High TMC polymers showed mechanical properties comparable to human arteries (Young’s moduli of 1.2–1.8 MPa and high elasticity with repeated cycling at 10% strain). Over 84 days degradation in PBS, the modulus and material strength decreased gradually. The polymers were nontoxic and showed good cell adhesion and proliferation over 7 days using human mesenchymal stem cells. When implanted into the rat peritoneal cavity, the polymers elicited formation of tissue capsules composed of myofibroblasts, resembling immature vascular smooth muscle cells. Thus, these polymers showed properties which were tunable and favorable for vascular tissue engineering, specifically, the growth of artificial blood vessels in vivo.

Sterilizing tissue-materials using pulsed power plasma

This paper investigates the potential of pulsed power to sterilize hard and soft tissues and its impact on their physico-mechanical properties. It hypothesizes that pulsed plasma can sterilize both vascular and avascular tissues and the transitive layers in between without deleterious effects on their functional characteristics. Cartilage/bone laminate was chosen as a model to demonstrate the concept, treated at low temperature, at atmospheric pressure, in short durations and in buffered environment using a purposed-built pulsed power unit. Input voltage and time of exposure were assigned as controlling parameters in a full factorial design of experiment to determine physical and mechanical alteration pre- and post-treatment. The results demonstrated that, discharges of 11 kV sterilized samples in 45 s, reducing intrinsic elastic modules from 1.4 ± 0.9 to 0.9 ± 0.6 MPa. There was a decrease of 14.1 % in stiffness and 27.8 % in elastic-strain energy for the top quartile. Mechanical impairment was directly proportional to input voltage (P value < 0.05). Bacterial inactivation was proportional to treatment time for input voltages above 32 V (P < 0.001; R Sq = 0.98). Thermal analysis revealed that helix-coil transition decelerated with exposure time and collagen fibrils were destabilized as denaturation enthalpy reduced by 200 μV. We concluded by presenting a safe operating threshold for pulsed power plasma as a feasible protocol for effective sterilization of connective tissues with varying level of loss in mechanical robustness which we argue to be acceptable in certain medical and tissue engineering application.

Species-specific homing mechanisms of human prostate cancer metastasis in tissue engineered bone

The development of effective therapeutic strategies against prostate cancer bone metastases has been impeded by the lack of adequate animal models that are able to recapitulate the biology of the disease in humans. Bioengineered approaches allow researchers to create sophisticated experimentally and physiologically relevant in vivo models to study interactions between cancer cells and their microenvironment under reproducible conditions. The aim of this study was to engineer a morphologically and functionally intact humanized organ bone which can serve as a homing site for human prostate cancer cells. Transplantation of biodegradable tubular composite scaffolds seeded with human mesenchymal progenitor cells and loaded with rhBMP-7 resulted in the development of a chimeric bone construct including a large number of human mesenchymal cells which were shown to be metabolically active and capable of producing extracellular matrix components. Micro-CT analysis demonstrated that the newly formed ossicle recapitulated the morphological features of a physiological organ bone with a trabecular network surrounded by a cortex-like outer structure. This microenvironment was supportive of the lodgement and maintenance of murine haematopoietic cell clusters, thus mimicking a functional organ bone. Bioluminescence imaging demonstrated that luciferase-transduced human PC3 cells reproducibly homed to the humanized tissue engineered bone constructs, proliferated, and developed macro-metastases. This model allows the analysis of interactions between human prostate cancer cells and a functional humanized bone organ within an immuno-incompetent murine host. The system can serve as a reproducible platform to study effects of therapeutics against prostate cancer bone metastases within a humanized microenvironment.

Mesenchymal stromal cells and the repair of cartilage tissue

Articular cartilage has a limited intrinsic repair capacity, and thus defects are more likely to further degrade rather than undergo spontaneous self-repair. Whilst a number of surgical techniques have been developed to repair cartilage defects, their efficacy is generally poor and total joint replacement remains the gold standard, albeit last resort, treatment option. Cell-based therapies hold the greatest promise, as they appear uniquely capable of generating de novo cartilage tissue. Two approved therapies (ACI and MACI) are based on the premise that the transplantation of ex vivo expanded autologous chondrocyte populations, harvested from a non-load bearing region of the same joint, could be utilized to effectively regenerate cartilage tissue in the primary defect site. These therapeutic strategies are partially limited by our inability to harvest and expand adequate numbers of autologous chondrocytes that retain the appropriate phenotype. By contrast, the harvest and expansion of large numbers of mesenchymal stem/stromal cells (MSC) derived from tissues such as bone marrow and adipose is comparatively straightforward and has become routine in laboratories worldwide. Additionally, our understanding of the biochemical and biophysical signals required to drive the chondrogenic differentiation of MSC is rapidly increasing. It is conceivable that in the near future MSC expansion and differentiation technologies will offer a means to generate sufficient cell numbers, of an appropriate phenotype, for use in cartilage defect repair. In this chapter we review the relative potential of MSC and their likely contribution to cartilage regeneration.

Mechanical tension as a driver of connective tissue growth in vitro

We propose the progressive mechanical expansion of cell-derived tissue analogues as a novel, growth-based approach to in vitro tissue engineering. The prevailing approach to producing tissue in vitro is to culture cells in an exogenous “scaffold” that provides a basic structure and mechanical support. This necessarily pre-defines the final size of the implantable material, and specific signals must be provided to stimulate appropriate cell growth, differentiation and matrix formation. In contrast, surgical skin expansion, driven by increments of stretch, produces increasing quantities of tissue without trauma or inflammation. This suggests that connective tissue cells have the innate ability to produce growth in response to elevated tension. We posit that this capacity is maintained in vitro, and that order-of-magnitude growth may be similarly attained in self-assembling cultures of cells and their own extracellular matrix. The hypothesis that growth of connective tissue analogues can be induced by mechanical expansion in vitro may be divided into three components: (1) tension stimulates cell proliferation and extracellular matrix synthesis; (2) the corresponding volume increase will relax the tension imparted by a fixed displacement; (3) the repeated application of static stretch will produce sustained growth and a tissue structure adapted to the tensile loading. Connective tissues exist in a state of residual tension, which is actively maintained by resident cells such as fibroblasts. Studies in vitro and in vivo have demonstrated that cellular survival, reproduction, and matrix synthesis and degradation are regulated by the mechanical environment. Order-of-magnitude increases in both bone and skin volume have been achieved clinically through staged expansion protocols, demonstrating that tension-driven growth can be sustained over prolonged periods. Furthermore, cell-derived tissue analogues have demonstrated mechanically advantageous structural adaptation in response to applied loading. Together, these data suggest that a program of incremental stretch constitutes an appealing way to replicate tissue growth in cell culture, by harnessing the constituent cells’ innate mechanical responsiveness. In addition to offering a platform to study the growth and structural adaptation of connective tissues, tension-driven growth presents a novel approach to in vitro tissue engineering. Because the supporting structure is secreted and organised by the cells themselves, growth is not restricted by a “scaffold” of fixed size. This also minimises potential adverse reactions to exogenous materials upon implantation. Most importantly, we posit that the growth induced by progressive stretch will allow substantial volumes of connective tissue to be produced from relatively small initial cell numbers.

Revascularization and tissue regeneration of an empty root canal space is enhanced by a direct blood supply and stem cells

Background Regenerative endodontics is an innovative treatment concept aiming to regenerate pulp, dentin and root structures. In the diseased or necrotic tooth, the limitation in vascular supply renders successful tissue regeneration/generation in a whole tooth challenging. The aim of this study is to evaluate the ability of vascularized tissue to develop within a pulpless tooth using tissue engineering techniques. Materials and methods A pulpless tooth chamber, filled with collagen I gel containing isolated rat dental pulp cells (DPC) and angiogenic growth factors, was placed into a hole created in the femoral cortex or into its own tooth socket, respectively. The gross, histological and biochemical characteristics of the de novo tissue were evaluated at 4 and 8weeks post-transplantation. Results Tooth revascularization and tissue generation was observed only in the femur group, confirming the important role of vascular supply in tissue regeneration. The addition of cells and growth factors significantly promoted connective tissue production in the tooth chamber. Conclusion Successful revascularization and tissue regeneration in this model demonstrate the importance of a direct vascular supply and the advantages of a stem cell approach. © 2012 John Wiley & Sons A/S.

Long-term stability of adipose tissue generated from a vascularized pedicled fat flap inside a chamber

Background Numerous studies demonstrate the generation and short-term survival of adipose tissue; however, long-term persistence remains elusive. This study evaluates long-term survival and transferability of de novo adipose constructs based on a ligated vascular pedicle and tissue engineering chamber combination. Methods Defined adipose tissue flaps were implanted into rats in either intact or perforated domed chambers. In half of the groups, the chambers were removed after 10 weeks and the constructs transferred on their vascular pedicle to a new site, where they were observed for a further 10 weeks. In the remaining groups, the tissue construct was observed for 20 weeks inside the chamber. Tissue volume was assessed using magnetic resonance imaging and histologic measures, and constructs were assessed for stability and necrosis. Sections were assessed histologically and for proliferation using Ki-67. Results At 20 weeks, volume analysis revealed an increase in adipose volume from 0.04 ± 0.001 ml at the time of insertion into the chambers to 0.27 ± 0.004 ml in the closed and 0.44 ± 0.014 ml in the perforated chambers. There was an additional increase of approximately 10 to 15 percent in tissue volume in flaps that remained in chambers for 20 weeks, whereas the volume of the transferred tissue not in chambers remained unaltered. Histomorphometric assessment of the tissues documented no signs of hypertrophy, fat necrosis, or atypical changes of the newly generated tissue. Conclusion This study presents a promising new method of generating significant amounts of mature, vascularized, stable, and transferable adipose tissue for permanent autologous soft-tissue replacement.

Long-term persistence of tissue-engineered adipose flaps in a murine model to 1 year : an update

BACKGROUND Tissue engineering of patient-specific adipose tissue has the potential to revolutionize reconstructive surgery. Numerous models have been described for the production of adipose tissue with success in the short term, but little has been reported on the stability of this tissue-engineered fat beyond 4 months. METHODS A murine model of de novo adipogenesis producing a potentially transplantable adipose tissue flap within 4 to 6 weeks was developed in the authors' laboratory. In this study, the authors assess the ability of three-chamber (44-μl volume) configurations shown to be adipogenic in previous short-term studies (autograft, n = 8; open, n = 6; fat flap, n = 11) to maintain their tissue volume for up to 12 months in vivo, to determine the most adipogenic configuration in the long term. RESULTS Those chambers having the most contact with existing vascularized adipose tissue (open and fat flap groups) showed increased mean adipose tissue percentage (77 ± 5.6 percent and 81 ± 6.9 percent, respectively; p < 0.0007) and volume (12 ± 6.8 μl and 30 ± 14 μl, respectively; p < 0.025) when compared with short-term controls and greater adipose tissue volume than the autograft (sealed) chamber group (4.9 ± 5.8 μl; p = 0.0001) at 1 year. Inclusion of a vascularized fat flap within the chamber produced the best results, with new fat completely filling the chamber by 1 year. CONCLUSIONS These findings demonstrate that fat produced by tissue engineering is capable of maintaining its volume when the appropriate microenvironment is provided. This has important implications for the application of tissue-engineering techniques in humans.

An arteriovenous loop in a protected space generates a permanent, highly vascular, tissue-engineered construct

A major obstacle to 3-dimensional tissue engineering is incorporation of a functional vascular supply to support the expanding new tissue. This is overcome in an in vivo intrinsic vascularization model where an arteriovenous loop (AVL) is placed in a noncollapsible space protected by a polycarbonate chamber. Vascular development and hypoxia were examined from 3 days to 112 days by vascular casting, morphometric, and morphological techniques to understand the model's vascular growth and remodeling parameters for tissue engineering purposes. At 3 days a fibrin exudate surrounded the AVL, providing a scaffold to migrating inflammatory, endothelial, and mesenchymal cells. Capillaries formed between 3 and 7 days. Hypoxia and cell proliferation were maximal at 7 days, followed by a peak in percent vascular volume at 10 days (23.20±3.14% compared with 3.59±2.68% at 3 days, P<0.001). Maximal apoptosis was observed at 112 days. The protected space and spontaneous microcirculatory development in this model suggest it would be applicable for in vivo tissue engineering. A temporal window in a period of intense angiogenesis at 7 to 10 days is optimal for exogenous cell seeding and survival in the chamber, potentially enabling specific tissue outcomes to be achieved.