Airway remodeling characteristic of asthma involves structural changes altering the elasticity of the airway smooth muscle (ASM) microenvironment potentially leading to ASM dysfunction. This effect of matrix stiffness was investigated using a physiologically relevant 3D culture model. Characterisation of microtissue responses with regards to contractile function and gene expression were studied varying the ECM stiffness and through stimulation with epithelial cell (AEC) conditioned media. ASM microtissues were fabricated under four different loading conditions and the matrix stiffness was increased by crosslinking through non-enzymatic glycation and increasing the collagen density. Function was assessed through the use of pharmacological agents and by imaging microcantilever deflection, used to calculate force generation. Crosslinking microtissues enhanced contractile function in response to agonists; however, this effect disappeared in microtissues tethered to stiff microcantilevers suggesting a limit of contractility within this model. Remarkably, there was a differential response in ASM function where increasing the collagen density (stiffness) significantly attenuated function. Additionally, contractility was significantly enhanced when chronically stimulated with AEC media. ASM tissue in 3D culture is responsive to the microenvironment stiffness and increases contractility in the presence of a stiffer ECM. This could occur with thickening of the airway wall in asthma. Decreased contractility with increased collagen density is in agreement with previous studies where it was shown that type I collagen is pro-proliferative and attenuates the contractile phenotype. We show the models ability to quantitatively demonstrate the impact of biomechanical cues on ASM function providing provides new ways to elucidate the mechanisms of cellular remodeling.

To obtain a cell source for cartilage tissue engineering primary cells are passaged on polystyrene dishes to increase cell number however, this stiff environment results in dedifferentiation. This study evaluates the role of microenvironment stiffness on regulation of passaged chondrocyte phenotype. Results show passaged cells on soft polyacrylamide gels (0.5kPa) become round, less proliferative, less contractile, have higher levels of globular actin (g-actin) compared to filamentous actin (f-actin), MRTF localization in the cytoplasm and down-regulation of MRTF associated genes such as type I collagen, alpha-smooth muscle actin, transgelin, tenascin C and vinculin. This suggests that the chondrogenic phenotype during passaging is regulated by actin polymerization and activation of MRTF signaling that induces expression of non-chondrogenic genes, and has functional effects as the cells become proliferative and contractile. Modulating substrate stiffness maybe a way to influence aspects of the chondrogenic phenotype in order to obtain sufficient cells suitable for cartilage tissue engineering.

Silicon substituted hydroxyapatite (SiHA) has been reported to produce faster bone in- growth in vitro compared to hydroxyapatite (HA). The mechanism by which silicate ions in these materials trigger bone growth and differentiation remains unclear. In vitro models were used in this thesis to investigate human osteoblast cell responses on exposure to silicon containing materials and silicate ion solutions. The amounts of serum protein bound to SiHA was significantly higher than that in HA (p<O.OOl). Culture of both primary human osteoblast (HOB) cells and an osteosarcoma cell line (MG-63 cells) showed that SiHA discs were biocompatible to the cells; flat cell morphologies, higher degree of cellular processes, and a covering with minuscule bone mineral-like crystals were observed. To elucidate the effects of silicate ions alone on osteoblast functions, a 1000 ppm standard silicon solution was supplemented into cell culture medium to produce silicate ion concentration of 20 and 500 ~M; it was found that the former had little effect on both cell types. Significant increases in levels of total DNA (p<0.001), protein (p<0.001), and collagen (p<0.001) were observed in HOB cells, but not in MG-63 cells, in cultures with 500 ~M silicate ions. Likewise, expression of COL-J al (p<0.001), BMP-2 (p<0.05), PHOSPHO-J (p<0.001) genes were up-regulated in both cells types cultured with 500 ~M silicate ions. Further studies proposed that the activation of cell proliferation by this silicate ion-containing medium, observed as increases in total DNA, involved TGF~1 and/or IGF-I receptors. In trying to understand this, it was latterly identified that the pH changes of the serum- supplemented culture medium that occurred during supplementation with the alkali silicate ion solution and subsequent neutralisation with HCI were the actual cause of the marked enhancement in HOB cell proliferation. Silicate ions did still appear to have a direct effect on some HOB cell responses, due to observing comparable effects of 20 and 500 ~M silicate ions on e.g. TNAP and PHOSPHO-J gene expression, compared to silicate ion-free controls.

Cardiovascular disease is the leading cause of death worldwide. Due to the extremely low natural regeneration rate of heart muscle, development of new therapeutics directed towards heart repair is challenging. A potential approach to regenerate damaged heart is offered by cardiac tissue engineering. Specifically, it aims at engineering cardiac muscle in vitro and implanting it into the site of injury so that it can be integrated into the host tissue and restore the heart’s function. To ensure the effectiveness of this technique, the engineered tissue needs to recapitulate structural and functional properties of the native myocardium. Myocardium consists of laminar sheets of uniaxially aligned cardiac muscle cells (cardiomyocytes) wrapped around the heart. Therefore, achieving high cardiomyocyte alignment in engineered muscle is crucial. In this study we aimed at stimulating cardiomyocyte alignment by mimicking their niche in the embryonic heart. We hypothesized that recapitulating the extracellular cues that guide myocardial development in the embryo can guide cardiac tissue organization in vitro. To test this hypothesis, we imaged the structure of fibronectin – the most abundant protein in embryonic heart’s extracellular matrix (ECM) – and derived a 2D pattern from it that was then microcontact printed onto a substrate to guide cell alignment. We compared chick cardiomyocyte alignment on the biomimetic pattern and line patterns that have been extensively studied in the past. Results revealed a unique cell density-dependent response of cardiomyocytes to the biomimetic pattern that allowed us to elucidate the role of cell-cell and cell-ECM interactions in cardiomyocyte alignment on fibronectin patterns by looking at the effect of local pattern features on alignment and inhibiting N-cadherin-based cell-cell junctions. Further, to engineer more clinically relevant tissues, we differentiated human induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) into cardiomyocytes and seeded them onto the fibronectin patterns. Cardiac tissues produced with these cells showed significant differences compared to the chick tissues due to their immature phenotype. We showed that co-culture with cardiac fibroblasts (CFBs) as well as maturation of iPSC-derived cardiomyocytes (iPSC-CMs) increased tissue alignment, indicating the important role of both of these factors in developing novel methods to engineer functional cardiac tissues.

Tendon, ligament, and joint capsular injuries represent 45% of the 32 million musculoskeletal injuries each year in the United States. Tendon injuries are especially common, requiring surgical repair for the shoulder’s rotator cuff tendons (51,000 per year), the Achilles tendon (44,000 per year), and the patellar tendon (42,000 per year). Tissue engineering provides an alternative in the treatment of tendon lesions through replacement of an injured tendon segment. The purpose of this study was to develop a tendon construct in vitro for clinical reconstructive surgery. Human tenocytes were isolated from hamstring tendons of patients who had undergone anterior cruciate ligament (ACL) surgeries. These tenocytes were cultured with culture media (α-MEM) supplemented with various concentrations of foetal bovine serum (FBS) (0%, 1%, 5% and 10%) and in the presence of different growth factors such as PDGFBB (0, 5, 10 and 50ng/ml), basic FGF (0, 5, 10 and 50ng/ml), IGF-1 (0, 10 and 50ng/ml) and TGFβ-3 (0, 1 and 10ng/ml). Fractional factorial design was utilized to select the combinations of growth factors that supported the following criteria: (1) the maximal cell proliferation with a minimum differentiation of the tenocytes in the presence of the least concentration of FBS possible and (2) maintaining cell survival and promoting tenocyte differentiation in FBS free culture media. The results have shown that: (i) The tenocyte cell number when cultured for 14 days in media supplemented with 1% FBS, 50ng/ml PDGFBB and 50ng/ml bFGF matched that of the positive control (10% FBS-treated cells). Not only was the collagen synthesis significantly reduced in these growth factor-treated cultures compared to positive control tenocytes, but also a significant inhibition of the mRNA expression of various tenocyte differentiation markers (Scleraxis, Tenomodulin, Collagen type I and Decorin) was evident. IGF-1 did not promote significant cell proliferation under low serum conditions but did induce tenocyte differentiation in vitro. Examination of the cell morphology confirmed that tenocytes were capable of less differentiation when cultured with 1% FBS, 50ng/ml PDGFBB and 50ng/ml bFGF, this culture condition was termed “the expansion phase”; (ii) The cell survival was maintained for up to 14 days in serum free culture media supplemented with 50ng/ml IGF-1 and 10ng/ml TGFβ-3 whilst cell differentiation was enhanced and evident by the increase in collagen synthesis and cell morphology. Furthermore, mRNA expression of the aforementioned cell differentiation markers were also significantly increased, this culture condition was termed “the differentiation phase”; (iii) By combining the culture condition optimized for the expansion and differentiation phase sequentially, it was possible to maintain a long term 2-D tenocyte culture in vitro for up to 28 days. In these cultures, the presence of dense collagen formation was clearly evident whereas in positive control group (10% FBS group) such observation was not noted even after prolonged culturing period of up to 45 days. These results suggested that the sequential treatment of tenocytes with growth factors identified for the expansion and differentiation phases was significantly more superior than the standard 10% FBS treatment; (iv) By combining the expansion and differentiation phases optimized for the 2-D cultures, it was possible to maintain human tenocytes in a 3-D scaffold (Bombix silk) for up to 28 days. The tendon like constructs that were formed, macroscopically and microscopically resembled the human hamstring tendon. This observation was confirmed by using H&E staining, scanning electron microscopy and by detecting collagen type I immunohistochemically; (v) It was possible to further validate these findings using in vivo animal models. This was undertaken by implanting the tenocytes cultured sequentially in the defined culture media described above, into the quadriceps of Balb/c nude male mice for up to 30 days. The nature and specificity of the tendon like structure that was formed after this implantation was investigated by H&E staining and immunohistochemistry. It was revealed that the culture conditions that were optimized during the expansion and differentiation phases were suitable for generating a human tendon reconstruct; a finding which is of significance due to its potential for tendon reconstructive surgery.

Electrospinning can be used to selectively process a variety of natural and synthetic polymers into highly porous scaffolds composed of nano-to-micron diameter fibers. This process shows great potential as a gateway to the development of physiologically relevant tissue engineering scaffolds. In this study we examine the structural and functional considerations regarding electrospun scaffolds for dermal template applications using novel quantification techniques. In order to characterize scaffold structure, a technique utilizing the fast Fourier transform was developed to systematically quantify fiber alignment and evaluate how different electrospinning parameters impact the structure and material properties of an electrospun scaffold. Gelatin was suspended at varying concentrations (80, 100, 130 and 150 mg/ml) and electrospun from 2,2,2 trifluoroethanol onto a rotating mandrel (200-7000 RPM). Scaffold anisotropy developed as a function of fiber diameter and mandrel speed and the induction of varying degrees of anisotropy imparted distinctive material properties to the electrospun scaffolds. Fiber alignment was the variable most closely associated with the regulation of peak stress, peak strain and modulus of elasticity. Next, we examined how the chemical and physical composition of the local microenvironment and the unmasking of possible RGD sensitive binding sites through collagen denaturation, independent of scaffold architecture and porosity, impacts cellular processes. We cultured human dermal fibroblasts on electrospun nylon coated with a variety of non-denatured and thermally denatured collagen-based proteins, as well as recovered electrospun collagen and gelatin (in an effort to examine if the electrospinning process degrades the collagen α chain). Differences in adhesion, proliferation and migration were exhibited between collagen-based proteins. Adhesion inhibition assays using a cyclic RGD peptide demonstrated no change in cell adhesion on non-denatured proteins and a significant drop in cell adhesion on thermally denatured proteins. Based on gel analysis and the results of our functional assays we conclude that collagen  chain structure is not directly altered by the electrospinning process. Overall, these results are critical to the understanding of how structure and architecture contribute to the overall properties of a scaffold, as well as how molecular variations can modulate scaffold functionality in a cellular environment.

In der Mund-, Kiefer- und Gesichtschirurgie besteht ein großer Bedarf an Transplantaten zur intra- und extraoralen Defektdeckung in der chirurgischen Therapie, insbesondere für die Rekonstruktion nach Traumen oder Tumorresektionen für den Erhalt von Funktion und Ästhetik. Konventionelle Methoden wie die Verwendung von autologen, freien Spalt- und Vollhaut-Transplantaten zeigen Nachteile wie z. B. die Entnahmemorbidität der Spenderregion oder die Notwendigkeit eines zweiten chirurgischen Eingriffs zur Deckung des Entnahmedefektes. Zudem sind diese Transplantate nur in kleinen Mengen verfügbar oder haben eine unterschiedliche Gewebestruktur sowie andere Keratinisierungsmuster. Diese Nachteile sollen mit Hilfe eines im Tissue Engineering hergestellten Oralmukosa-Äquivalentes umgangen werden. Dazu wurden zunächst Methoden zur Isolierung und Kultivierung primärer, oraler Fibroblasten bzw. Keratinozyten entwickelt, die das Ausgangsmaterial für die Herstellung von Dermal-Äquivalenten bzw. von organotypischen Kokulturen in vitro bilden. Die Zellen wurden sowohl histologisch als auch immunhistochemisch charakterisiert und nach Optimierung der Kulturbedingungen zur Entwicklung von Oralmukosa-Äquivalenten (OMÄs) eingesetzt. Dabei ist auch die Wahl eines geeigneten Trägermaterials ein entscheidender Faktor. Deshalb wurden in dieser Arbeit verschiedene Unterlagen auf Eignung als Scaffold für das Tissue Engineering von Oralmukosa getestet. Unter anderem wurden die Materialien Vicryl (resorbierbares Polyglactin-910-Netz), DRT (dermale Regenerationsmatrix aus bovinem Kollagen-I vernetzt mit einem Glycosaminoglycan) und TFE (equine Kollagen-I-Membran) in Zellkulturversuchen auf Biokompatibilität und Stabilität geprüft. Dazu wurden zunächst Fibroblasten auf die Scaffolds ausgesät um Dermal-Äquivalente (DÄs) zu erhalten. Das Wachstum der Zellen wurde mittels Elektronenmikroskopie sowie immunhistochemischen Methoden untersucht. Die Analyse zeigte gutes Zellwachstum und somit gute Biokompatibilität auf allen verwendeten Materialien. In folgenden Experimenten wurden zusätzlich Keratinozyten auf DÄs ausgesät und somit organotypische OMÄs entwickelt. Die generierten Konstrukte wurden mit Hilfe von IIF-Färbungen von Kryoschnitten sowie RT-qPCR bezüglich ihrer Zellarchitektur, ihrer Fähigkeit zur Bildung einer Basalmembran und ihrer Fähigkeit zur Differenzierung untersucht. Es stellte sich heraus, dass auf allen drei Trägern Fibroblasten-Keratinozyten Kulturen hergestellt werden konnten. Dabei zeigte Vicryl eine gute Biostabilität, jedoch ohne Ausbildung der natürlichen Stratifizierung der Keratinozytenschichten. Auf TFE dagegen zeigte sich die beste Architektur und Proliferation der Zellen mit Stratifizierung der Keratinozyten, allerdings eine schlechte Biostabilität. DRT stellte sich als die Matrix heraus, die die gewünschten Eigenschaften am besten vereint. Das Ergebnis war jedoch im Bezug auf die Dicke der Epithelschicht sowie deren Differenzierung und Ausbildung einer Basalmembran noch zu verbessern. Dies konnte mit Hilfe der Kulturmethode an der Luft-Flüssigkeits-Grenzfläche erreicht werden. Jedoch gelang bezüglich der Zellarchitektur noch immer kein optimales Ergebnis. Erst der Einsatz einer weiteren Membran, SIS (azellularisierter Schweinedarm), die durch ihren natürlichen Ursprung ähnlich strukturiert ist wie humane Submukosa, zeigte, dass die angewandte Methodik zur Herstellung von OMÄs funktionierte. Auf diesem Träger gelang die Herstellung eines Transplantates, das eine mit normaler Oralmukosa vergleichbare, reguläre Zellarchitektur mit dermaler und epidermaler Komponente aufwies, die qualitativ noch besser war als auf TFE. Auch die Biostabilität während des Versuchszeitraumes war wie bei Vicryl und DRT gegeben. Die Neusynthese einer Basalmembran konnte mittels IIF-Färbung nachgewiesen werden. Die Proliferation der Keratinozyten war in der Basalschicht lokalisiert und nahm Richtung apikal ab. Lediglich eine Differenzierung des Transplantates war mittels immunhistochemischer Methoden nicht nachweisbar. Auf diese Weise konnte in der vorliegenden Arbeit ein OMÄ entwickelt werden, dessen Aufbau mit dem von natürlicher Oralmukosa vergleichbar war. Die in dieser Arbeit gewonnen Erkenntnisse dienen somit als Grundlage zur Optimierung und Verwirklichung des klinischen Einsatzes von mittels Tissue Engineering hergestellten, autologen OMÄs. / In oral and maxillofacial surgery, there is a great demand for oral mucosa equivalents for intra- and extraoral grafting as oral reconstruction after trauma or tumor resections to preserve function and aesthetics. Current methods of covering intraoral defects with autologous epidermal and dermal grafts show disadvantages, e.g. donor site morbidity or the need for a second surgical procedure to cover the harvesting site. These grafts are either only available in small amounts or have a different texture, such as a different keratinization pattern. These disadvantages could be avoided by using autologous tissue engineered mucosa equivalents. In a first step, a method for isolation and cultivation of primary oral fibroblasts and keratinocytes was developed. They formed the starting material for production of dermal equivalents (DEs) and organotypic cocultures in vitro. The cells were characterized both by histological staining and by immunohistochemical staining. After optimization of the cell culture conditions they were used for the development of oral mucosa equivalents (OMEs). Thereby the selection of a suitable scaffold is a decisive factor. Therefore, different matrices for the tissue engineering of oral mucosa have been studied in this thesis. Amongst others, the materials Vicryl (woven membrane of polyglactin 910), DRT (dermal regeneration template of cross-linked bovine tendon collagen and a glycosaminoglycan) and TFE (equine collagen I membrane) have been tested in cell-culture experiments for biocompatibility and stability. For this purpose first only fibroblasts were seeded on scaffolds to obtain DEs. Cell growth was examined by electron microscopy and immunohistochemical methods. The analysis showed good cell growth and good biocompatibility as well on all the used materials. In following experiments keratinocytes were seeded on dermal equivalents to develop organotypic co-cultures. The obtained constructs were characterized by immunohistochemical staining and gene expression analysis using RT-qPCR to get information about cell architecture, formation of a basal membrane and status of differentiation. It has been found, that it was possible to create fibroblast-keratinocyte-cultures on all three scaffolds. Thereby Vicryl showed a good biostability but no formation of the natural stratification of the epidermal cells. These findings are in contrast to the results on TFE. Here was the best architecture and proliferation of the cells with stratification of the newly formed epidermis, but bad biostability of the membrane. The combination of the desired properties could only be seen on DRT, but the thickness, differentiation and basal membrane formation of the epithelial layer needed to be improved. This was achieved by cultivation of the cells in the air liquid interface but there was still no optimal result. Only the use of another scaffold, SIS (acellular matrix from porcine small bowel) which has a similar structure to humane submucosa, shows functionality of the developed method of OME generation. On this scaffold the generation of a transplant succeeded even better than on TFE. It had dermal and epidermal components with regular cell architecture like native oral mucosa. Also the biostability during the experimental period was comparable with Vicryl and DRT. It has been possible to demonstrate the formation of a basal membrane by IIF staining. Keratinocyte proliferation was localized in the basal layer with declining proliferation activity in apical direction. Only differentiation could not be proven by means of immunohistochemistry. Thus the present thesis developed a method for creation of an OME with comparable organization to that of the native oral mucosa. The findings summarized in this study will serve as basis for optimization and realization of the clinical use of tissue engineered autologous OMEs.

Diffusive mass transport is fundamental for many scientific research areas including physics, chemistry, biology, pharmacy, medicine and geography. In tissue engineering and regenerative medicine, the diffusive mass transport property of artificial and natural biological materials is a key parameter for understanding 3D scaffolds towards designing vascular networks capable of mimicking natural tissues. The aim was to understand diffusion coefficient differences for biomedical materials of different geometrical shapes and matrix properties, including collagen gels and polymeric membranes. Theoretical work involved producing analytical expressions for diffusion, variously in a planar sheet, a cylinder and a sphere for different initial and boundary conditions. Dynamic amperometric current responses at recessed, membrane covered planar and hanging mercury drop electrodes were also studied. Experimentally, glucose and lactate needle enzyme electrodes were fabricated and an experimental rig was designed to measure analyte concentrations within gels. The analyte diffusion coefficient in a collagen gel was obtained by fitting the simulated to the experimental concentration profiles. Also, a membrane covered planar electrode system was developed to measure the diffusion coefficient of electrochemically active solute through various polymeric barriers. Here, a fit of the simulated to the experimental amperometric current transients was made. Conventionally, a drug release curve is used to characterise drug release, which depends on drug concentration and substrate geometric size and shape. A more intrinsic property, the effective diffusion coefficient, independent of drug concentration or substrate, was determined by fitting calculated drug release to experimental curves. Finally, solute diffusion across dual laminar flows in a microfluidic system was analysed and used to determine ammonia diffusion coefficient in aqueous solution. The key novelty of this work was the construction of a series of accurate but simple expressions for mass transport in various geometric matrices which enabled the determination of diffusion coefficients by a specific analytical expression obtained from Fick’s Laws and the best fit, avoiding extensive numerical computation such as finite element methods. For all the above, corresponding one point equations were also derived to give initial rapid estimates of diffusion coefficients.

The application of prolonged mechanical loading to cartilage alters the osmolality of the extracellular environment, with osmotic challenge known to alter the gene expression and the metabolic activity of chondrocytes. However, the mechanisms by which osmolality controls chondrocyte activity remain unclear. Previous study on various cell types, including chondrocytes, showed that hyper-osmotic challenge induces the condensation of chromatin, with highly condensed chromatin often associated with gene poor regions of DNA and gene silencing. The present study investigated the effect of osmotic challenge on chromatin organisation, genome wide gene-expression and the cellular and nuclear deformability of chondrocytes. In order to observe a broad effect of osmotic challenge on the nuclei, the chondrocytes were subjected to a range of hypo- and hyper-osmotic challenge and imaged by confocal microscopy. Chromatin condensation was quantified by the Sobel edge algorithm in MATLAB. Hyper-osmotic challenge on chondrocytes induced an increase in chromatin condensation. Interestingly, the most marked condensation occurred within the osmolality range of articular cartilage in vivo. The effect of osmotic challenge varied between the monolayer cultured and agarose seeded chondrocytes, which may be due to the differences in cytoskeleton organisation between the two culture conditions. Additionally, chromatin condensation induced by hyper-osmotic challenge was shown to be reversible. Marked differences were observed in the deformability of the cell and nucleus in chondrocytes post osmotic challenge, compared to the 300 mOsm/kg conditions typically used for in vitro isolated chondrocyte studies. From the microarray study, the application of 500 mOsm/kg for both 1 and 5 hours altered the gene expression, including the expression of histone related genes, with a higher number of genes affected by the 5 hours hyper-osmotic challenge. The findings of this study suggest that osmotically-induced alterations in nuclei morphology and chromatin structure may provide a direct biophysical mechanism that controls chondrocytes activity.

The anterior cruciate ligament (ACL) is a major ligament within the knee joint. Its role is to provide stability and maintain the physiological kinetics and kinematics of the joint. ACL injuries are common as a result of sporting and traffic accidents and current therapeutic options do not fully restore the joint kinetics and kinematics. As such, patients often suffer from increased joint laxity and joint pain following an ACL reconstruction and this can lead to secondary problems such as osteoarthritis. It is believed that improving the ACL graft could help restore the normal kinetics and kinematics of the knee joint and hence postpone or prevent the onset of primary and secondary problems. Tissue engineering has the potential to provide functional tissue to repair or replace injured or diseased tissues in the patient. The ACL is a tissue which could benefit from such developments and thus improve the success of the reconstruction. However, the ACl is a complex structure made up of a highly orientated collagen hierarchy which experiences three dimensional loading in vivo. For an engineered tissue to be functional it is necessary for this orientated structure to be replicated. The appropriate structure is achieved by replication of the in vivo ACL strain pattern which requires combined tensile and torsional loading. Current custommade and commercially available bioreactors have not been able to fully replicate this motion with the necessary feedback and monitoring of mechanical parameters. The aim of this project was to develop a novel bioreactor with physiological mechanical conditioning for the tissue engineering of an anterior cruciate ligament. A bioreactor capable of applying complex tensile and torsional loading to a developing ACL was designed, manufactured and validated. The bioreactor which has been developed is a novel research tool which allows the effect of a number of parameters to be investigated in a 3D loading environment. It can be used for the engineering of connective tissues such as ligaments and tendons and has the potential to be adapted for use with other musculoskeletal tissues such as bone. It could also be used for research to understand the processes involved in the growth and development of tissues.