Biomimetic grafts from ultrafine fibers for collagenous tissues.

BACKGROUND: The ligament is the soft tissue that connects bone to bone and, in case of severe injury or rupture, it cannot heal itself mainly because of its poor vascularity and dynamic nature. Tissue engineering carries the potential to restore the injured tissue functions by utilization of scaffolds mimicking the structure of native ligament. Collagen fibrils in the anterior cruciate ligament (ACL) have a diameter ranging from 20 to 300 nm, which defines the physical and mechanical properties of the tissue. Also, the ACL tissue exhibited a bimodal distribution of collagen fibrils. Currently, the ability to fabricate scaffolds replicating this structure is a significant challenge. OBJECTIVE: This work aims at i) measuring the diameter of collagens of bovine ACL tissue, ii) investigating the fabrication of sub-100 nm fibers, and iii) fabricating aligned scaffolds with bimodal diameter distribution (with two peaks) resembling the healthy ACL structure. It is hypothesized that such scaffolds can be produced by electrospinning polycaprolactone (PCL) solutions. METHODS: To test the hypothesis, various PCL solutions were formulated in acetone and formic acid in combination with pyridine, and electrospun to generate sub-100 nm fibers. Next, this formulation was adjusted to produce nanofibers with a diameter between 100 nm and 200 nm. Finally, these solutions were combined in the co-electrospinning process, i.e., two-spinneret electrospinning, to fabricate biomimetic scaffolds with a bimodal distribution. RESULTS: Electrospinning of 8% and 15% PCL solutions, respectively, resulted in the production of fibers with diameters below and above 100 nm. The combined scaffold exhibited a bimodal distribution of aligned fibers with peaks around 80 and 180 nm, thus mimicking the collagen fibrils of healthy ACL tissue. CONCLUSION: This research is expected to have a society-wide impact because it aims to enhance the health condition and life quality of a wide range of patients.

Braided biomimetic PCL grafts for anterior cruciate ligament repair and regeneration.

Anterior cruciate ligament (ACL) is a knee joint stabilizer with a limited regeneration capacity mainly because of low cellular content. State-of-the-art procedures are unable to restore the functions of the tissue as demonstrated by limited success rates. Regenerative engineering can offer a solution for restoring the functions of torn/ruptured ligaments provided that biomimetic grafts are available as grafts/scaffolds. However, a model construct to test behavior of cells to better understand the healing mechanism of ACL is still missing. This study, firstly, aimed at creating an injured rabbit ACL model. Then, the injured and healthy ACL tissues were characterized in terms of alignment and diameter distributions of collagen fibrils. Next, polycaprolactone (PCL) grafts were prepared from braided electrospun meshes and were characterized in terms of alignment and diameter distributions of fibers. Finally, biomechanical properties of ACL tissue and mechanical properties of PCL grafts were determined and compared. Findings demonstrated that distributions of the fiber diameters of PCL electrospun grafts were similar to diameter distribution of collagens of healthy and injured rabbit ACL. The novelty of this study relies on the determination of the diameter distribution of collagens of healthy and injured rabbit ACL tissues, and fabrication of PCL grafts with diameter distributions similar to that seen in healthy and injured ACLs. This study is significant because it addresses a worldwide clinical problem associated with millions of patients. The fibrous biomimetic graft designed in this study is different from the traditional grafts that exhibit unimodal distribution, and it is expected to have a significant contribution to ACL regeneration efforts.

Osteochondral Interface: Regenerative Engineering and Challenges.

Osteochondral (OC) defects are debilitating for patients and represent a significant clinical problem for orthopedic surgeons as well as regenerative engineers due to their potential complications, which are likely to lead to osteoarthritis and related diseases. If they remain untreated or are treated suboptimally, OC lesions are known to impact the articular cartilage and the transition from cartilage to bone, that is, the cartilage-bone interface. An important component of the OC interface, that is, a selectively permeable membrane, the tidemark, still remains unaddressed in more than 90% of the published research in the past decade. This review focuses on the structure, composition, and function of the OC interface, regenerative engineering attempts with different scaffolding strategies and challenges ahead of us in recapitulating the native OC interface. There are different schools of thought regarding the structure of the native OC interface: stratified and graded. The former assumes the cartilage-to-bone interface to be hierarchically divided into distinct yet continuous zones of uncalcified cartilage-calcified cartilage-subchondral bone. The latter assumes the interface is continuously graded, that is, formed by an infinite number of layers. The cellular composition of the interface, either in respective layers or continuously changing in a graded manner, is chondrocytes, hypertrophic chondrocytes, and osteoblasts as moved from cartilage to bone. Functionally, the interface is assumed to play a role in enabling a smooth transition of loads exerted on the cartilage surface to the bone underneath. Regenerative engineering involves, first, a characterization of the native OC interface in terms of the composition, structure, and function, and, then, proposes the appropriate biomaterials, cells, and biomolecules either alone or in combination to eventually form a structure that mimics and functionally behaves similar to the native interface. The major challenge regarding regeneration of the OC interface appears to lie, in addition to others, in the formation of tidemark, which is a thin membrane separating the OC interface into two distinct zones: the avascular OC interface and the vascular OC interface. There is a significant amount of literature on regenerative approaches to the OC interface; however, only a small portion of them consider the importance of tidemark. Therefore, this review aims at highlighting the significance of the structural organization of the components of the OC interface and increasing the awareness of the orthopedics community regarding the importance of tidemark formation after clinical interventions or regenerative engineering attempts.

Collagen Fibril Diameter Distribution of Sheep Anterior Cruciate Ligament.

The anterior cruciate ligament (ACL) tissue is a soft tissue connecting the femur and tibia at the knee joint and demonstrates a limited capacity for self-regeneration due to its low vascularity. The currently available clinical procedures are unable to fully restore damaged ACL tissue, and tissue engineering can offer options with a potential of restoring the torn/ruptured ACL by using biomimetic constructs that are similar to native tissue in terms of structure, composition, and functions. However, a model substrate to understand how the ACL cells regenerate the injured tissue is still not available. In this study, it is hypothesized that the nanofiber-based model substrate with bimodal and unimodal fiber diameter distributions will mimic the diameter distribution of collagen fibrils seen in healthy and injured sheep ACL, respectively. The aims were to (i) create an ACL injury in a sheep ACL by applying extensional force to rupture the healthy ACL tissue, (ii) measure the collagen fibril diameter distributions of healthy and injured ACL, (iii) fabricate polycaprolactone (PCL) nanofiber-based model constructs using electrospinning with diameter distributions similar to healthy and injured ACL tissue, and (iv) measure mechanical properties of ACL tissue and PCL electrospun constructs. The results showed that the fiber diameter distributions of PCL electrospun constructs and those of the healthy and injured ACL tissues were similar. The novelty in this investigation is that the collagen fibril diameter distribution of healthy and injured sheep ACL tissues was reported for the first time. The study is significant because it aims to create a model construct to solve an important orthopedic-related clinical problem affecting millions of people globally. The model construct fabricated in this work is expected to have an important impact on ACL regeneration efforts.

A biomimetic synthetic nanofiber-based model for anterior cruciate ligament regeneration.

Reconstructed ACL cannot completely restore its functions due to absence of physiologically viable environment for optimal biomaterial-cell interaction. Currently available procedures only mechanically attach grafts to bone without any biological integration. How the ACL cells perform this biological attachment is not fully understood partly due to the absence of appropriate environment to test cell behavior both in vitro and in vivo. Availability of biomimetic models would enable the scientists to better explore the behavior of cells at health and during tissue healing. In this study, it is hypothesized that the collagen fibril diameter distribution in rat ACL changes from a bimodal distribution in the healthy ACL to a unimodal distribution after injury, and that this change can be mimicked in synthetic nanofiber-based constructs. This hypothesis was tested by first creating an injured rat ACL model by applying a mechanical tensile force to the healthy ACL tissue until rupture. Secondly, the collagen fibril diameter distributions of healthy and injured ACL tissue were determined, and polycaprolactone (PCL) constructs were created to mimic the distributions of collagen fibrils in healthy and injured tissues. Findings reveal that the fiber diameter distribution of aligned bimodal PCL constructs were similar to that of the collagen fibrils in native ACL tissue. This study is significant because suggested bimodal and unimodal fibrous model constructs, respectively, represent a healthy and injured tissue environment and the behavior of ACL cells cultured on these constructs may provide significant input on ACL regeneration mechanism.

Change in Collagen Fibril Diameter Distribution of Bovine Anterior Cruciate Ligament upon Injury Can Be Mimicked in a Nanostructured Scaffold.

More than 200,000 people are suffering from Anterior Cruciate Ligament (ACL) related injuries each year in the US. There is an unmet clinical demand for improving biological attachment between grafts and the host tissue in addition to providing mechanical support. For biological graft integration, it is important to provide a physiologically feasible environment for the host cells to enable them to perform their duties. However, behavior of cells during ACL healing and the mechanism of ACL healing is not fully understood partly due to the absence of appropriate environment to test cell behavior both in vitro and in vivo. This study aims at (i) investigating the change in fibril diameter of bovine ACL tissue upon injury and (ii) fabricating nanofiber-based scaffolds to represent the morphology and structure of healthy and injured ACL tissues. We hypothesized that distribution and mean diameter of ACL fibrils will be altered upon injury. Findings revealed that the collagen fibril diameter distribution of bovine ACL changed from bimodal to unimodal upon injury with subsequent decrease in mean diameter. Polycaprolactone (PCL) scaffold fiber diameter distribution exhibited similar bimodal and unimodal distribution behavior to qualitatively represent the cases of healthy and injured ACL, respectively. The native ACL tissue demonstrated comparable modulus values only with the aligned bimodal PCL scaffolds. There was significant difference between mechanical properties of aligned bimodal and unaligned unimodal PCL scaffolds. We believe that the results obtained from measurements of diameter of collagen fibrils of native bovine ACL tissue can serve as a benchmark for scaffold design.

A bioartificial rat heart tissue: Perfusion decellularization and characterization.

Despite remarkable advancement in the past decades, heart-related defects are still prone to progress irreversibly and can eventually lead to heart failure. A personalized extracellular matrix-based bioartificial heart created by allografts/xenografts emerges as an alternative as it can retain the original three-dimensional architecture combined with a preserved natural heart extracellular matrix. This study aimed at developing a procedure for decellularizing heart tissue harvested from rats and evaluating decellularization efficiency in terms of residual nuclear content and structural properties. Tissue sections showed no or little visible cell nuclei in decellularized heart, whereas the native heart showed dense cellularity. In addition, there was no significant variation in the alignment of muscle fibers upon decellularization. Furthermore, no significant difference was detected between native and decellularized hearts in terms of fiber diameter. Our findings demonstrate that fiber alignment and diameter can serve as additional parameters in the characterization of biological heart scaffolds as these provide valuable input for evaluating structural preservation of decellularized heart. The bioartificial scaffold formed here can be functionalized with patient's own material and utilized in regenerative engineering.

Effect of gadolinium concentration on temperature change under magnetic field.

Gadolinium based contrast agents (GBCAs) were found to play a role in nephrogenic systemic fibrosis in patients with and without renal impairment. Therefore, preserving the structural stability of GBCAs to reduce their propensity to liberate Gd3+ is of utmost importance. This study evaluates the effect of gadolinium concentration of GBCAs on solution temperature under magnetic fields. It is hypothesized that presence of gadolinium will lead to temperature changes of its solutions under magnetic field, and this change will depend on concentration. In this study, GBCAs were diluted to concentrations of 0.6, 1.2, 1.8, 2.4 mMol/L. A 10mL preparation in pure water, simulated body fluid (SBF), and plasma was scanned at 3T following a soft tissue neck protocol, and their temperatures were measured. Findings revealed that concentration of GBCA had significant effect on temperature change in all dilution media. Type of commercially available GBCA had an effect only in SFB and plasma. Evaluation of correlation between conditional stability constant (Kcond) and temperature difference (ΔT) revealed that in water and SBF there exists a positive correlation between Kcond and temperature variation. Collectively, GBCAs can cause local temperature variations when administered into patients, and can affect dissociation of gadolinium from its chelates, which should be investigated in a further study.

3D-printed poly(lactic acid) scaffolds for trabecular bone repair and regeneration: scaffold and native bone characterization.

PURPOSE: Study objectives were set to (i) fabricate 3D-printed scaffolds/grafts with varying pore sizes, (ii) characterize surface and mechanical properties of scaffolds, (iii) characterize biomechanical properties of bovine trabecular bone, and (iv) evaluate attachment and proliferation of human bone marrow mesenchymal stem cells on 3D-printed scaffolds. MATERIALS AND METHODS: Poly(lactic acid) scaffolds were fabricated using 3D-printing technology, and characterized in terms of their surface as well as compressive mechanical properties. Trabecular bone specimens were obtained from bovine and characterized biomechanically under compression. Human bone marrow mesenchymal stem cells were seeded on the scaffolds, and their attachment capacity and proliferation were evaluated. RESULTS: Contact angles and compressive moduli of scaffolds decreased with increasing pore dimensions of 0.5 mm, 1.0 mm, and 1.25 mm. Biomechanical characterization of trabecular bone yielded higher modulus values as compared to scaffolds with all pore sizes studied. Human bone marrow mesenchymal stem cells attached to the surfaces of all scaffolds yet proliferated more on scaffolds with 1.25 mm pore size. CONCLUSIONS: Collectively, given the similarity between 3D-printed scaffolds and native bone in terms of pore size, porosity, and appropriate mechanical properties of scaffolds, the 3D-printed poly(lactic acid) (PLA) scaffolds of this study appear as candidate substitutes for bone repair and regeneration.

Polymer fiber-based models of connective tissue repair and healing.

Physiologically relevant models of wound healing are essential for understanding the biology of connective tissue repair and healing. They can also be used to identify key cellular processes and matrix characteristics critical for the design of soft tissue grafts. Modeling the various stages of repair post tendon injury, polymer meshes of varying fiber diameter (nano-1 (390 nm) < nano-2 (740 nm) < micro (1420 nm)) were produced. Alignment was also introduced in the nano-2 group to model matrix undergoing biological healing rather than scar formation. The response of human tendon fibroblasts on these model substrates were evaluated over time as a function of fiber diameter and alignment. It was observed that the repair models of unaligned nanoscale fibers enhanced cell growth and collagen synthesis, while these outcomes were significantly reduced in the mature repair model consisting of unaligned micron-sized fibers. Organization of paxillin and actin on unaligned meshes was enhanced on micro- compared to nano-sized fibers, while the expression and activity of RhoA and Rac1 were greater on nanofibers. In contrast, aligned nanofibers promoted early cell organization, while reducing excessive cell growth and collagen production in the long term. These results show that the early-stage repair model of unaligned nanoscale fibers elicits a response characteristic of the proliferative phase of wound repair, while the more mature model consisting of unaligned micron-sized fibers is more representative of the remodeling phase by supporting cell organization while suppressing growth and biosynthesis. Interestingly, introduction of fiber alignment in the nanofiber model alters fibroblast response from repair to healing, implicating matrix alignment as a critical design factor for circumventing scar formation and promoting biological healing of soft tissue injuries.

Synthesis and characterization of polycaprolactone for anterior cruciate ligament regeneration.

Anterior cruciate ligament (ACL) is the most frequently torn ligament in the knee, and complete healing is unlikely due to lack of vascularization. Current approaches for the treatment of ACL injuries include surgical interventions and grafting, however recent reports show that surgeries have 94% recurrency, and that repaired tissues are biomechanically inferior to the native tissue. These necessitate the need for new strategies for scar-free repair/regeneration of ACL injuries. Polycaprolactone (PCL) is a biodegradable and biocompatible synthetic polymer, which has been widely used in the connective tissue repair/regeneration attempts. Here, we report on the synthesis of PCL via ring opening polymerization using ε-caprolactone as the monomer, and ammonium heptamolybdate as a catalyst. The synthesized PCL was characterized using Fourier Transform Infrared Spectroscopy (FTIR) and Nuclear Magnetic Resonance (NMR) spectroscopy. It was then processed using electrospinning to form nanofiber-based scaffolds. These scaffolds were characterized in terms of surface as well as mechanical properties, and compared to the properties of commercially available PCL, and of native ACL tissue harvested from sheep. In addition, scaffolds fabricated with synthesized PCL were evaluated regarding their cell attachment capacity using human bone marrow mesenchymal stem cells (hBMSCs). Our findings demonstrated that the synthesized PCL is similar to its commercially available counterpart in terms of surface morphology and mechanical properties. In addition, fibrous scaffolds generated with electrospinning showed weaker mechanical properties visa vis native ACL tissue in terms of ultimate stress, and elastic modulus. Also, the synthesized PCL can accommodate cell attachment when tested with hBMSCs. Putting together, these observations reveal that the PCL synthesized in this study could be a good candidate as a biomaterial for ligament repair or regeneration.

Effects of QMix and ethylenediaminetetraacetic acid on decalcification and erosion of root canal dentin.

The aim of this study was to evaluate the effect of initial NaOCl on the decalcification and erosion ability of EDTA and QMix. Sixty-maxillary-incisors were bisected longitudinally and the tooth-halves were used. The experiment was conducted in two-sets. In set-I, 80-tooth halves were treated in the presence or absence of initial NaOCl and EDTA. In set-II, 40-tooth halves were immersed in NaOCl and QMix. After each treatment, calcium-ion release was determined with flame photometry. The erosion was imaged using SEM. Initial NaOCl led to concentration- and time-dependent increase in calcium removal effect of 17% EDTA (p < .05). The rate of calcium removal and root canal wall erosion was considerably more severe with the use of 5% NaOCl for 3 min (p < .05). QMix as a final solution showed less decalcification and erosion than 17% EDTA when used 5% NaOCl as an initial irrigant (p < .05). Optimizing the concentration and application time of NaOCl can decrease the decalcification effect of chelating agents.

Characterization of Human Dental Pulp Tissue Under Oscillatory Shear and Compression.

Availability of material as well as biological properties of native tissues is critical for biomaterial design and synthesis for regenerative engineering. Until recently, selection of biomaterials and biomolecule carriers for dental pulp regeneration has been done randomly or based on experience mainly due to the absence of benchmark data for dental pulp tissue. This study, for the first time, characterizes the linear viscoelastic material functions and compressive properties of human dental pulp tissue harvested from wisdom teeth, under oscillatory shear and compression. The results revealed a gel-like behavior of the pulp tissue over the frequency range of 0.1-100 rps. Uniaxial compression tests generated peak normal stress and compressive modulus values of 39.1 ± 20.4 kPa and 5.5 ± 2.8 kPa, respectively. Taken collectively, the linear viscoelastic and uniaxial compressive properties of the human dental pulp tissue reported here should enable the better tailoring of biomaterials or biomolecule carriers to be employed in dental pulp regeneration.

Viscoelastic Properties of Dental Pulp Tissue and Ramifications on Biomaterial Development for Pulp Regeneration.

INTRODUCTION: A critical step in biomaterial selection effort is the determination of material as well as the biological properties of the target tissue. Previously, the selection of biomaterials and carriers for dental pulp regeneration has been solely based on empirical experience. METHODS: In this study, first, the linear viscoelastic material functions and compressive properties of miniature pig dental pulp were characterized using small-amplitude oscillatory shear and uniaxial compression at a constant rate. They were then compared with the properties of hydrogels (ie, agarose, alginate, and collagen) that are widely used in tissue regeneration. RESULTS: The comparisons of the linear viscoelastic material functions of the native pulp tissue with those of the 3 hydrogels revealed the gel-like behavior of the pulp tissue over a relatively large range of time scales (ie, over the frequency range of 0.1-100 rps). At the constant gelation agent concentration of 2%, the dynamic properties (ie, storage and loss moduli and the tanδ) of the collagen-based gel approached those of the native tissue. Under uniaxial compression, the peak normal stresses and compressive moduli of the agarose gel were similar to those of the native tissue, whereas alginate and collagen exhibited significantly lower compressive properties. CONCLUSIONS: The linear viscoelastic and uniaxial compressive properties of the dental pulp tissue reported here should enable the more appropriate selection of biogels for dental pulp regeneration via the better tailoring of gelation agents and their concentrations to better mimic the dynamic and compressive properties of native pulp tissue.

Protein-releasing polymeric scaffolds induce fibrochondrocytic differentiation of endogenous cells for knee meniscus regeneration in sheep.

Regeneration of complex tissues, such as kidney, liver, and cartilage, continues to be a scientific and translational challenge. Survival of ex vivo cultured, transplanted cells in tissue grafts is among one of the key barriers. Meniscus is a complex tissue consisting of collagen fibers and proteoglycans with gradient phenotypes of fibrocartilage and functions to provide congruence of the knee joint, without which the patient is likely to develop arthritis. Endogenous stem/progenitor cells regenerated the knee meniscus upon spatially released human connective tissue growth factor (CTGF) and transforming growth factor-ß3 (TGFß3) from a three-dimensional (3D)-printed biomaterial, enabling functional knee recovery. Sequentially applied CTGF and TGFß3 were necessary and sufficient to propel mesenchymal stem/progenitor cells, as a heterogeneous population or as single-cell progenies, into fibrochondrocytes that concurrently synthesized procollagens I and IIα. When released from microchannels of 3D-printed, human meniscus scaffolds, CTGF and TGFß3 induced endogenous stem/progenitor cells to differentiate and synthesize zone-specific type I and II collagens. We then replaced sheep meniscus with anatomically correct, 3D-printed scaffolds that incorporated spatially delivered CTGF and TGFß3. Endogenous cells regenerated the meniscus with zone-specific matrix phenotypes: primarily type I collagen in the outer zone, and type II collagen in the inner zone, reminiscent of the native meniscus. Spatiotemporally delivered CTGF and TGFß3 also restored inhomogeneous mechanical properties in the regenerated sheep meniscus. Survival and directed differentiation of endogenous cells in a tissue defect may have implications in the regeneration of complex (heterogeneous) tissues and organs.

Scaffold fiber diameter regulates human tendon fibroblast growth and differentiation.

The diameter of collagen fibrils in connective tissues, such as tendons and ligaments is known to decrease upon injury or with age, leading to inferior biomechanical properties and poor healing capacity. This study tests the hypotheses that scaffold fiber diameter modulates the response of human tendon fibroblasts, and that diameter-dependent cell responses are analogous to those seen in healthy versus healing tissues. Particularly, the effect of the fiber diameter (320 nm, 680 nm, and 1.80 µm) on scaffold properties and the response of human tendon fibroblasts were determined over 4 weeks of culture. It was observed that scaffold mechanical properties, cell proliferation, matrix production, and differentiation were regulated by changes in the fiber diameter. More specifically, a higher cell number, total collagen, and proteoglycan production were found on the nanofiber scaffolds, while microfibers promoted the expression of phenotypic markers of tendon fibroblasts, such as collagen I, III, V, and tenomodulin. It is possible that the nanofiber scaffolds of this study resemble the matrix in a state of injury, stimulating the cells for matrix deposition as part of the repair process, while microfibers represent the healthy matrix with micron-sized collagen bundles, thereby inducing cells to maintain the fibroblastic phenotype. The results of this study demonstrate that controlling the scaffold fiber diameter is critical in the design of scaffolds for functional and guided connective tissue repair, and provide new insights into the role of matrix parameters in guiding soft tissue healing.

Biomimetic scaffold design for functional and integrative tendon repair.

Rotator cuff tears represent the most common shoulder injuries in the United States. The debilitating effect of this degenerative condition coupled with the high incidence of failure associated with existing graft choices underscores the clinical need for alternative grafting solutions. The 2 critical design criteria for the ideal tendon graft would require the graft to not only exhibit physiologically relevant mechanical properties but also be able to facilitate functional graft integration by promoting the regeneration of the native tendon-to-bone interface. Centered on these design goals, this review will highlight current approaches to functional and integrative tendon repair. In particular, the application of biomimetic design principles through the use of nanofiber- and nanocomposite-based scaffolds for tendon tissue engineering will be discussed. This review will begin with nanofiber-based approaches to functional tendon repair, followed by a section highlighting the exciting research on tendon-to-bone interface regeneration, with an emphasis on implementation of strategic biomimicry in nanofiber scaffold design and the concomitant formation of graded multi-tissue systems for integrative soft-tissue repair. This review will conclude with a summary and discussion of future directions.

Osteochondral tissue formation through adipose-derived stromal cell differentiation on biomimetic polycaprolactone nanofibrous scaffolds with graded insulin and Beta-glycerophosphate concentrations.

The ability to fabricate tissue engineering scaffolds containing systematic gradients in the distributions of stimulators provides additional means for the mimicking of the important gradients observed in native tissues. Here the concentration distributions of two bioactive agents were varied concomitantly for the first time (one increasing, whereas the other decreasing monotonically) in between the two sides of a nanofibrous scaffold. This was achieved via the application of a new processing method, that is, the twin-screw extrusion and electrospinning method, to generate gradients of insulin, a stimulator of chondrogenic differentiation, and ß-glycerophosphate (ß-GP), for mineralization. The graded poly(ɛ-caprolactone) mesh was seeded with human adipose-derived stromal cells and cultured over 8 weeks. The resulting tissue constructs were analyzed for and revealed indications of selective differentiation of human adipose-derived stromal cells toward chondrogenic lineage and mineralization as functions of position as a result of the corresponding concentrations of insulin and ß-GP. Chondrogenic differentiation of the stem cells increased at insulin-rich locations and mineralization increased at ß-GP-rich locations.

Viscoelastic and biomechanical properties of osteochondral tissue constructs generated from graded polycaprolactone and beta-tricalcium phosphate composites.

The complex micro-/nanostructure of native cartilage-to-bone insertion exhibits gradations in extracellular matrix components, leading to variations in the viscoelastic and biomechanical properties along its thickness to allow for smooth transition of loads under physiological movements. Engineering a realistic tissue for osteochondral interface would, therefore, depend on the ability to develop scaffolds with properly graded physical and chemical properties to facilitate the mimicry of the complex elegance of native tissue. In this study, polycaprolactone nanofiber scaffolds with spatially controlled concentrations of beta-tricalcium phosphate nanoparticles were fabricated using twin-screw extrusion-electrospinning process and seeded with MC3T3-E1 cells to form osteochondral tissue constructs. The objective of the study was to evaluate the linear viscoelastic and compressive properties of the native bovine osteochondral tissue and the tissue constructs formed in terms of their small-amplitude oscillatory shear, unconfined compression, and stress relaxation behavior. The native tissue, engineered tissue constructs, and unseeded scaffolds exhibited linear viscoelastic behavior for strain amplitudes less than 0.1%. Both native tissue and engineered tissue constructs demonstrated qualitatively similar gel-like behavior as determined using linear viscoelastic material functions. The normal stresses in compression determined at 10% strain for the unseeded scaffold, the tissue constructs cultured for four weeks, and the native tissue were 0.87+/-0.08 kPa, 3.59+/-0.34 kPa, and 210.80+/-8.93 kPa, respectively. Viscoelastic and biomechanical properties of the engineered tissue constructs were observed to increase with culture time reflecting the development of a tissuelike structure. These experimental findings suggest that viscoelastic material functions of the tissue constructs can provide valuable inputs for the stages of in vitro tissue development.

Functionally graded electrospun polycaprolactone and beta-tricalcium phosphate nanocomposites for tissue engineering applications.

Fabricating functionally graded scaffolds from biodegradable polymers to enable the mimicking of native tissue is an important challenge. Here we demonstrate the fabrication and utilization of functionally graded non-woven meshes of polycaprolactone incorporated with tricalcium phosphate nanoparticles using a new hybrid twin-screw extrusion/electrospinning (TSEE) process, which allows the time-dependent feeding of various solid and liquid ingredients and their melting, dispersion, deaeration and pressurization together with electrospinning within the confines of a single process. Using this hybrid method, the concentration of tricalcium phosphate nanoparticles could be tailored to vary in a targeted/controlled manner between the two surfaces of the scaffold mesh. The graded scaffolds were seeded and cultured with mouse preosteoblast cells (MC3T3-E1). Within 4 weeks, the tissue constructs revealed the formation of continuous gradations in extracellular matrix with various markers including collagen synthesis and mineralization, akin to the type of variations observed in the typical bone-cartilage interface in terms of the distributions of concentration of Ca particles and of mechanical properties associated with this. The demonstrated hybrid method should allow much better control of the distributions of various ingredients, including the concentrations of drugs/growth factors, as well as the porosity, mechanical property, wettability, biodegradation rate distributions in tissue engineering scaffolds, aiming to mimic the elegant complex distributions found in native tissue.