Collagen Microstructure Research Articles

Matrix architecture plays a pivotal role in 3D osteoblast migration: The effect of interstitial fluid flow.

Osteoblast migration is a crucial process in bone regeneration, which is strongly regulated by interstitial fluid flow. However, the exact role that such flow exerts on osteoblast migration is still unclear. To deepen the understanding of this phenomenon, we cultured human osteoblasts on 3D microfluidic devices under different fluid flow regimes. Our results show that a slow fluid flow rate by itself is not able to alter the 3D migratory patterns of osteoblasts in collagen-based gels but that at higher fluid flow rates (increased flow velocity) may indirectly influence cell movement by altering the collagen microstructure. In fact, we observed that high fluid flow rates (1 µl/min) are able to alter the collagen matrix architecture and to indirectly modulate the migration pattern. However, when these collagen scaffolds were crosslinked with a chemical crosslinker, specifically, transglutaminase II, we did not find significant alterations in the scaffold architecture or in osteoblast movement. Therefore, our data suggest that high interstitial fluid flow rates can regulate osteoblast migration by means of modifying the orientation of collagen fibers. Together, these results highlight the crucial role of the matrix architecture in 3D osteoblast migration. In addition, we show that interstitial fluid flow in conjunction with the matrix architecture regulates the osteoblast morphology in 3D.

Stiffness of Cell Micro-Environment Guides Long Term Cell Growth in Cell Seeded Collagen Microspheres

Mesenchymal stem cells are widely implicated as a cell source for tissue engineering of skeletal tissue in cell-based therapy. Physical and mechanical cues are potent controlling factors in cell differentiation and can be implemented as a guide to study cellular response, matrix production and tissue regeneration. Microspheres were produced by gelation of bovine collagen type I with concentration of 2 mg/mL and 1,000-2,000 cells per droplet. Short and long term cell viability of human embryonic stem cell-derived mesenchymal progenitors (hES-MPs) and MG-63 osteoblastic cells as well as collagen microstructure and contraction were monitored during 28 days post encapsulation (pc). Results indicated that collagen concentration, hence mechanical properties of cell’s extracellular micro-environment are important in cell proliferation and differentiation. Contraction of cell-embedded microspheres was found to be vital in cell adaptation and the remodelling of their new environment. It was also found that collagen concentration of 2 mg/mL supports proliferation of hES-MPs while higher collagen concentration promoted the viability of MG-63s. Results of hES-MPs characterization in 3D soft environment and mechanically stimulated hES-MPs collagen microspheres can be used in cells/therapeutic carriers, implants in bone and cartilage healing applications. The microspheres developed in this study can also be used as a tool to build more optimised construct to transfer mechanically stimulated stem cells to the specific area of a defective bone which would add significant benefit to the field of bone regeneration.

\u03bcMAPPS: a novel phasor approach to second harmonic analysis for in vitro-in vivo investigation of collagen microstructure

Second Harmonic Generation (SHG) is a label-free imaging method used to monitor collagen organization in tissues. Due to its sensitivity to the incident polarization, it provides microstructural information otherwise unreachable by other intensity based imaging methods. We develop and test a Microscopic Multiparametric Analysis by Phasor projection of Polarization-dependent SHG (μMAPPS) that maps the features of the collagen architecture in tissues at the micrometer scale. μMAPPS retrieves pixel-by-pixel the collagen fibrils anisotropy and orientation by operating directly on two coupled phasor spaces, avoiding direct fitting of the polarization dependent SHG signal. We apply μMAPPS to fixed tissue sections and to the study of the collagen microscopic organization in tumors ex-vivo and in-vivo. We develop a clustering algorithm to automatically group pixels with similar microstructural features. μMAPPS can perform fast analyses of tissues and opens to future applications for in-situ diagnosis of pathologies and diseases that could assist histo-pathological evaluation.

3D collagen fibrillar microstructure guides pancreatic cancer cell phenotype and serves as a critical design parameter for phenotypic models of EMT.

Pancreatic cancer, one of the deadliest cancers, is characterized by high rates of metastasis and intense desmoplasia, both of which are associated with changes in fibrillar type I collagen composition and microstructure. Epithelial to mesenchymal transition (EMT), a critical step of metastasis, also involves a change in extracellular matrix (ECM) context as cells detach from basement membrane (BM) and engage interstitial matrix (IM). The objective of this work was to develop and apply an in-vitro three-dimensional (3D) tumor-ECM model to define how ECM composition and biophysical properties modulate pancreatic cancer EMT. Three established pancreatic ductal adenocarcinoma (PDAC) lines were embedded within 3D matrices prepared with type I collagen Oligomer (IM) at various fibril densities to control matrix stiffness or Oligomer and Matrigel combined at various ratios while maintaining constant matrix stiffness. Evaluation of cell morphology and protein expression at both the cellular- and population-levels revealed a spectrum of matrix-driven EMT phenotypes that were dependent on ECM composition and architecture as well as initial PDAC phenotype. In general, exposure to fibrillar IM was sufficient to drive EMT, with cells displaying spindle-shaped morphology and mesenchymal markers, and non-fibrillar BM promoted more epithelial behavior. When cultured within low density Oligomer, only a subpopulation of epithelial BxPC-3 cells displayed EMT while mesenchymal MiaPaCa-2 cells displayed more uniform spindle-shaped morphologies and mesenchymal marker expression. Interestingly, as IM fibril density increased, associated changes in spatial constraints and matrix stiffness resulted in all PDAC lines growing as tight clusters; however mesenchymal marker expression was maintained. Collectively, the comparison of these results to other in-vitro tumor models highlights the role of IM fibril microstructure in guiding EMT heterogeneity and showcases the potential of standardized 3D matrices such as Oligomer to serve as robust platforms for mechanistic study of metastasis and creation of predictive drug screening models.

Mechanical Characterization and Material Modeling of Diabetic Aortas in a Rabbit Model.

Diabetes has been recognized as a major risk factor to cause macrovascular diseases and plays a key role in aortic wall remodeling. However, the effects of diabetes on elastic properties of aortas remain largely unknown and quantitative mechanical data are lacking. Thirty adult rabbits (1.6-2.2kg) were collected and the type 1 diabetic rabbit model was induced by injection of alloxan. A total of 15 control and 15 diabetic rabbit (abdominal) aortas were harvested. Uniaxial and biaxial tensile tests were performed to measure ultimate tensile strength and to characterize biaxial mechanical behaviors of the aortas. A material model was fitted to the biaxial experimental data to obtain constitutive parameters. Histological and mass fraction analyses were performed to investigate the underlying microstructure and dry weight percentages of elastin and collagen in the control and the diabetic aortas. No statistically significant difference was found in ultimate tensile strength between the control and the diabetic aortas. Regarding biaxial mechanical responses, the diabetic aortas exhibited significantly lower extensibility and significantly higher tissue stiffness than the control aortas. Notably, tissue stiffening occurred in both circumferential and axial directions for the diabetic aortas; however, mechanical anisotropy does not change significantly. The material model was able to fit biaxial experimental data very well. Histology showed that a number of isolated foam cells were embedded in the diabetic aortas and hyperplasia of collagen was identified. The dry weight percentages of collagen within the diabetic aortas increased significantly as compared to the control aortas, whereas no significant change was found for that of elastin. Our data suggest that the diabetes impairs elastic properties and alters microstructure of the aortas and consequently, these changes may further contribute to complex aortic wall remodeling.

Multiscale biphasic modelling of peritumoural collagen microstructure: The effect of tumour growth on permeability and fluid flow.

We present an in-silico model of avascular poroelastic tumour growth coupled with a multiscale biphasic description of the tumour–host environment. The model is specified to in-vitro data, facilitating biophysically realistic simulations of tumour spheroid growth into a dense collagen hydrogel. We use the model to first confirm that passive mechanical remodelling of collagen fibres at the tumour boundary is driven by solid stress, and not fluid pressure. The model is then used to demonstrate the influence of collagen microstructure on peritumoural permeability and interstitial fluid flow. Our model suggests that at the tumour periphery, remodelling causes the peritumoural stroma to become more permeable in the circumferential than radial direction, and the interstitial fluid velocity is found to be dependent on initial collagen alignment. Finally we show that solid stresses are negatively correlated with peritumoural permeability, and positively correlated with interstitial fluid velocity. These results point to a heterogeneous, microstructure-dependent force environment at the tumour–peritumoural stroma interface.

Trauma induces overexpression of Cx43 in human fetal membrane defects.

ObjectiveWe developed an in vitro model to examine whether trauma induces connexin 43 (Cx43) expression and collagen organisation in the amniotic membrane (AM) of fetal membrane (FM) defects.MethodTerm human FM was traumatised in vitro. Cell morphology and Cx43 were examined in the wound edge AM by immunofluorescence (IMF) confocal microscopy and compared to control AM. Collagen microstructure was examined by second harmonic generation (SHG) imaging. Cell viability was assessed with calcein and ethidium staining.ResultsAfter trauma, the AM showed a dense region of cells, which had migrated towards the wound edge. In wound edge AM, Cx43 puncta was preferentially distributed in mesenchymal cells compared to epithelial cells with significant expression in the fibroblast layer than epithelial layer (p < 0.001). In the fibroblast layer, the collagen fibres were highly polarised and aligned in parallel to the axis of the wound edge AM. There was an absence of cell migration across the defect with no healing after 168 h. Cell viability of the FM after trauma was maintained during culture.ConclusionCx43 overexpression in wounded AM drives structural changes in collagen that slows down efficacy of cell migration across the FM defect. © 2017 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd.

The collagen microstructural changes of rat menisci and tibiofemoral cartilages under the influence of mechanical loading: An in vitro wear test of whole joints.

Knee osteoarthritis (OA) is suggested to be induced by multi-factors, and mechanical environment is regarded as a risky factor. To investigate the effect of isolated mechanical factor on cartilage. An active wear test system was designed to perform parameters-controlled in vitro wear tests on rat knee joints with specific load magnitude, flexion-extension angle, and movement frequency. Six hind limbs of 9-month-old male Sprague-Dawley rats, with an additional spring on the medial side, were worn by using the custom-designed apparatus. Researchers observed both the menisci and tibial cartilages of these hind limbs using multiphoton laser scanning microscopy to analyze the change of the collagen microstructure caused by wear. Collagen microstructure of both the medial and lateral meniscus became disordered under cyclic load. Some tissues on the surface of the medial tibial cartilage were removed and the middle layer of the medial compartment displayed cracks. On the contrary, the lateral tibial cartilage was intact. The results implied that cyclic load caused menisci microstructure disarrangement prior to tibial cartilage damage and the collagen structure of mid-layer tibial cartilage failed before that of the superficial layer under the kinematics adopted in the study.

Biaxial loading of arterial tissues with 3D in situ observations of adventitia fibrous microstructure: A method coupling multi-photon confocal microscopy and bulge inflation test

Disorders in the wall microstructure underlie all forms of vascular disease, such as the aortic aneurysm, the rupture of which is necessarily triggered at the microscopic level. In this context, we developed an original experimental approach, coupling a bulge inflation test to multiphoton confocal microscopy, for visualizing the 3D micro-structure of porcine, human non-aneurysmal and aneurysmal aortic adventitial collagen under increasing pressurization. The experiment complexity on such tissues led to deeply address the acquisition major hurdles. The important innovative features of the methodology are presented, especially regarding region-of-interest tracking, definition of a stabilization period prior to imaging and correction of z-motion, z being the objective's axis. Such corrections ensured consistent 3D qualitative and quantitative analyses without z-motion. Qualitative analyses of the stable 3D images showed dense undulated collagen fiber bundles in the unloaded state which tended to progressive straightening and separation into a network of thinner bundles at high pressures. Quantitative analyses were made using a combination of weighted 2D structure tensors and fitting of 4 independent Gaussian functions to measure parameters related to straightening and orientation of the fibers. They denoted 3 principal fibers directions, approximately 45°, 135° and 90° with respect to the circumferential axis in the circumferential-axial plane without any evident reorientation of the fibers under pressurization. Results also showed that fibers at zero-pressure state were straighter and less dispersed in orientation for human samples – especially aneurysms – than for pigs. Progressive straightening and decrease in dispersion were quantified during the inflation. These findings provide further insight into the micro-architectural changes within the arterial wall.

Development of overuse tendinopathy: A new descriptive model for the initiation of tendon damage during cyclic loading.

Tendinopathic tissue has long been characterized by changes to collagen microstructure. However, initial tendon damage from excessive mechanical loading-a hallmark of tendinopathy development-could occur at the nanoscale level of collagen fibrils. Indeed, it is on this scale that tenocytes interact directly with tendon matrix, and excessive collagen fibril damage not visible at the microscale could trigger a degenerative cascade. In this study, we explored whether initiation of tendon damage during cyclic loading occurs via a longitudinal compression-induced buckling mechanism of collagen fibrils leading to nanoscale kinkband development. Two groups of tendons were cyclically loaded to equivalent peak stresses. In each loading cycle, tendons in one group were unloaded to the zero displacement mark, while those in the other group were unloaded to a nominal level of tension, minimizing the potential for fibril buckling. Tendons that were unloaded to the zero displacement mark ruptured significantly sooner during cyclic loading (1,446 ± 737 vs. 4,069 ± 1,129 cycles), indicating that significant fatigue damage is accrued in the low stress, toe region of the load-deformation response. Ultrastructural analysis using scanning electron microscopy of tendons stopped after 1,000 cycles showed that maintaining a nominal tension slowed the accumulation of kinkbands, supporting a longitudinal compression-induced buckling mechanism as the basis for kinkband development. Based on our results, we present a new descriptive model for the initiation of tendon damage during cyclic loading. The so-called Compression of Unrecovered Elongation or CUE Model may provide useful insight into the development of tendinopathy. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 36:467-476, 2018.

Pneumatically Actuated Soft Micromold Device for Fabricating Collagen and Matrigel Microparticles.

Collagen microparticles have recently gained more attention as viable cell confinement blocks in many biomedical research fields. Small volume and high surface area of collagen structure improve cell confinement, viability, and proliferation. Moreover, dense collagen fiber structure can protect cells from immune destruction. The ability to produce collagen microparticles in an accurate and reliable way is of upmost importance to the advancement of many biomedical researches, especially cancer research and tissue engineering. Currently, no such fabrication technique exists due to inherent fragility of collagen. Herein, we report the very first platform, pneumatically actuated soft micromold (PASMO) device, which addresses challenges in collagen microparticle production. Our new platform uses a soft micromold with a pneumatic actuator that can produce arbitrary shapes of collagen microstructures precisely from 100 μm to over 2 mm in range and can encapsulate cells inside without damaging the shape. The duplication accuracy of more than 96% in dimensions and 90% in depth has been demonstrated. The density of collagen fiber distribution is determined to be 86.57%, which is higher than that of collagen microparticles produced by other methods. We have confirmed cell viability in collagen microparticles. We also produce Matrigel™ particles as tool to develop a xenograft cancer model. The results demonstrate that Matrigel particles created by the PASMO device can reduce cell scattering for the xenograft model and the uniformity of tumors developed in mice is 12-fold improved, which can lead to an increased accuracy of cancer metastasis studies and drug screening research. These breakthroughs in the production of modular microparticles will push the boundaries of cancer research in the near future.

Effects of collagen microstructure and material properties on the deformation of the neural tissues of the lamina cribrosa.

The loss of visual function associated with glaucoma has been attributed to sustained mechanical insult to the neural tissues of the lamina cribrosa due to elevated intraocular pressure. Our study is the first computational model built from specimen-specific tissue microstructure to consider the mechanics of the neural tissues of the lamina separately from the connective tissue. We found that the deformation of the neural tissue was much larger than that predicted by any recent microstructure-aware models of the lamina. These results are consistent with recent experimental data and the highest deformations were found in the region of the lamina where glaucomatous damage first occurs. This study provides new insight into the complex biomechanical environment within the lamina.

Ultrasound exposure during collagen polymerization produces pro-migratory fiber structures

Non-invasive techniques to control protein structure and function are needed for tissue engineering applications. This study tested the hypothesis that non-thermal effects of ultrasound can produce changes in collagen microstructure that support directional cell migration. Type I rat tail collagen was polymerized for 15 minutes in the presence of an 8-MHz ultrasound standing wave field over a range of 0 to 30 W/cm2 spatial peak, temporal average intensity. Temperature-matched sham gels were manufactured in a heated water bath without exposure to ultrasound. To test for effects of acoustically-modified collagen on cellular behavior, fibronectin-null mouse embryonic fibroblasts or mouse skin explants were seeded on gel surfaces and cultured up to 7 days prior to imaging with phase or second harmonic generation (SHG) microscopy. Acellular acoustically-modified collagen gels were characterized by regions of radial fiber alignment, increased pore size, and denser fibers, with greater heterogeneity at higher intensities. Ultrasound-exposed gels supported rapid directional cell migration with accumulation of cells in the regions of highest SHG signal. Neither fiber alignment nor cellular migration was observed in temperature-matched sham gels. Results demonstrate that ultrasound exposure during collagen polymerization can result in functionally altered collagen microstructure, in part through a non-thermal mechanism.

High-resolution study of the 3D collagen fibrillary matrix of Achilles tendons without tissue labelling and dehydrating.

Knowledge of the collagen structure of an Achilles tendon is critical to comprehend the physiology, biomechanics, homeostasis and remodelling of the tissue. Despite intensive studies, there are still uncertainties regarding the microstructure. The majority of studies have examined the longitudinally arranged collagen fibrils as they are primarily attributed to the principal tensile strength of the tendon. Few studies have considered the structural integrity of the entire three-dimensional (3D) collagen meshwork, and how the longitudinal collagen fibrils are integrated as a strong unit in a 3D domain to provide the tendons with the essential tensile properties. Using second harmonic generation imaging, a 3D imaging technique was developed and used to study the 3D collagen matrix in the midportion of Achilles tendons without tissue labelling and dehydration. Therefore, the 3D collagen structure is presented in a condition closely representative of the in vivo status. Atomic force microscopy studies have confirmed that second harmonic generation reveals the internal collagen matrix of tendons in 3D at a fibril level. Achilles tendons primarily contain longitudinal collagen fibrils that braid spatially into a dense rope-like collagen meshwork and are encapsulated or wound tightly by the oblique collagen fibrils emanating from the epitenon region. The arrangement of the collagen fibrils provides the longitudinal fibrils with essential structural integrity and endows the tendon with the unique mechanical function for withstanding tensile stresses. A novel 3D microscopic method has been developed to examine the 3D collagen microstructure of tendons without tissue dehydrating and labelling. The study also provides new knowledge about the collagen microstructure in an Achilles tendon, which enables understanding of the function of the tissue. The knowledge may be important for applying surgical and tissue engineering techniques to tendon reconstruction.

Cell-instructive starPEG-heparin-collagen composite matrices.

Cell-instructive materials play an important role in the generation of both regenerative therapies and advanced tissue and disease models. For that purpose, biofunctional polymer hydrogels recapitulating molecular cues of the extracellular matrix (ECM) were successfully applied in various different studies. However, hydrogels generally lack the hierarchical supramolecular structure of natural ECM. We have therefore developed a hydrogel composite material made from starPEG-peptide conjugates, maleimide-functionalized heparin and collagen type I fibrils. The collagen-laden scaffolds showed a heterogeneous local variation in the stiffness of the material. The composite gels were successfully tested in culture experiments with human umbilical cord vein endothelial cells and bone marrow-derived human mesenchymal stem cells. It was concluded that the composite scaffold was able to faithfully mimic important cell-instructive features of the ECM.

Inverted orientation improves decellularization of whole porcine hearts.

The use of decellularized tissue as a suitable scaffold for engineered tissue has emerged over the past decade as one of the most promising biofabrication platforms. The decellularization process removes all native cells, leaving the natural biopolymers, extracellular matrix materials and native architecture intact. This manuscript describes heart orientation as important in optimizing decellularization efficiency and maintaining extracellular matrix integrity. To our knowledge, this is the first study to assess flow dynamics in a whole heart throughout the decellularization procedure. Our findings compared to currently published methods demonstrate that continuous complex real-time measurements and analyses are required to produce an optimal scaffold for cardiac regeneration.

Imaging of Scleral Collagen Deformation Using Combined Confocal Raman Microspectroscopy and Polarized Light Microscopy Techniques.

This work presents an optospectroscopic characterization technique for soft tissue microstructure using site-matched confocal Raman microspectroscopy and polarized light microscopy. Using the technique, the microstructure of soft tissue samples is directly observed by polarized light microscopy during loading while spatially correlated spectroscopic information is extracted from the same plane, verifying the orientation and arrangement of the collagen fibers. Results show the response and orientation of the collagen fiber arrangement in its native state as well as during tensile and compressive loadings in a porcine sclera model. An example is also given showing how the data can be used with a finite element program to estimate the strain in individual collagen fibers. The measurements demonstrate features that indicate microstructural reorganization and damage of the sclera’s collagen fiber arrangement under loading. The site-matched confocal Raman microspectroscopic characterization of the tissue provides a qualitative measure to relate the change in fibrillar arrangement with possible chemical damage to the collagen microstructure. Tests and analyses presented here can potentially be used to determine the stress-strain behavior, and fiber reorganization of the collagen microstructure in soft tissue during viscoelastic response.

Microstructure and mechanics of healthy and aneurysmatic abdominal aortas: experimental analysis and modelling

Soft biological tissues such as aortic walls can be viewed as fibrous composites assembled by a ground matrix and embedded families of collagen fibres. Changes in the structural components of aortic walls such as the ground matrix and the embedded families of collagen fibres have been shown to play a significant role in the pathogenesis of aortic degeneration. Hence, there is a need to develop a deeper understanding of the microstructure and the related mechanics of aortic walls. In this study, tissue samples from 17 human abdominal aortas (AA) and from 11 abdominal aortic aneurysms (AAA) are systematically analysed and compared with respect to their structural and mechanical differences. The collagen microstructure is examined by analysing data from second-harmonic generation imaging after optical clearing. Samples from the intact AA wall, their individual layers and the AAA wall are mechanically investigated using biaxial stretching tests. A bivariate von Mises distribution was used to represent the continuous fibre dispersion throughout the entire thickness, and to provide two independent dispersion parameters to be used in a recently proposed material model. Remarkable differences were found between healthy and diseased tissues. The out-of-plane dispersion was significantly higher in AAA when compared with AA tissues, and with the exception of one AAA sample, the characteristic wall structure, as visible in healthy AAs with three distinct layers, could not be identified in AAA samples. The collagen fibres in the abluminal layer of AAAs lost their waviness and exhibited rather straight and thick struts of collagen. A novel set of three structural and three material parameters is provided. With the structural parameters fixed, the material model was fitted to the mechanical experimental data, giving a very satisfying fit although there are only three material parameters involved. The results highlight the need to incorporate the structural differences into finite-element simulations as otherwise simulations of AAA tissues might not be good predictors for the actual in vivo stress state.

Creation of an injectable in situ gelling native extracellular matrix for nucleus pulposus tissue engineering

Background ContextDisc degeneration is the leading cause of low back pain and is often characterized by a loss of disc height, resulting from cleavage of chondroitin sulfate proteoglycans (CSPGs) present in the nucleus pulposus. Intact CSPGs are critical to water retention and maintenance of the nucleus osmotic pressure. Decellularization of healthy nucleus pulposus tissue has the potential to serve as an ideal matrix for tissue engineering of the disc because of the presence of native disc proteins and CSPGs. Injectable in situ gelling matrices are the most viable therapeutic option to prevent damage to the anulus fibrosus and future disc degeneration. PurposeThe purpose of this research was to create a gentle decellularization method for use on healthy nucleus pulposus tissue explants and to develop an injectable formulation of this matrix to enable therapeutic use without substantial tissue disruption. Study DesignPorcine nuclei pulposi were isolated, decellularized, and solubilized. Samples were assessed to determine the degree of cell removal, matrix maintenance, gelation ability, cytotoxic residuals, and native cell viability. MethodsNuclei pulposi were decellularized using serial detergent, buffer, and enzyme treatments. Decellularized nuclei pulposi were solubilized, neutralized, and buffered. The efficacy of decellularization was assessed by quantifying DNA removal and matrix preservation. An elution study was performed to confirm removal of cytotoxic residuals. Gelation kinetics and injectability were quantified. Long-term in vitro experiments were performed with nucleus pulposus cells to ensure cell viability and native matrix production within the injectable decellularized nucleus pulposus matrices. ResultsThis work resulted in the creation of a robust acellular matrix (>96% DNA removal) with highly preserved sulfated glycosaminoglycans (>47%), and collagen content and microstructure similar to native nucleus pulposus, indicating preservation of disc components. Furthermore, it was possible to create an injectable formulation that gelled in situ within 45 minutes and formed fibrillar collagen with similar diameters to native nucleus pulposus. The processing did not result in any remaining cytotoxic residuals. Solubilized decellularized nucleus pulposus samples seeded with nucleus pulposus cells maintained robust viability (>89%) up to 21 days of culture in vitro, with morphology similar to native nucleus pulposus cells, and exhibited significantly enhanced sulfated glycosaminoglycans production over 21 days. ConclusionsA gentle decellularization of porcine nucleus pulposus followed by solubilization enabled the creation of an injectable tissue-specific matrix that is well tolerated in vitro by nucleus pulposus cells. These matrices have the potential to be used as a minimally invasive nucleus pulposus therapeutic to restore disc height.

Effect of crosslinking in cartilage-like collagen microstructures

The mechanical performance of biological tissues is underpinned by a complex and finely balanced structure. Central to this is collagen, the most abundant protein in our bodies, which plays a dominant role in the functioning of tissues, and also in disease. Based on the collagen meshwork of articular cartilage, we have developed a bottom-up spring-node model of collagen and examined the effect of fibril connectivity, implemented by crosslinking, on mechanical behaviour. Although changing individual crosslink stiffness within an order of magnitude had no significant effect on modelling predictions, the density of crosslinks in a meshwork had a substantial impact on its behaviour. Highly crosslinked meshworks maintained a ‘normal’ configuration under loading, with stronger resistance to deformation and improved recovery relative to sparsely crosslinked meshwork. Stress on individual fibrils, however, was higher in highly crosslinked meshworks. Meshworks with low numbers of crosslinks reconfigured to disease-like states upon deformation and recovery. The importance of collagen interconnectivity may provide insight into the role of ultrastructure and its mechanics in the initiation, and early stages, of diseases such as osteoarthritis.