Evaluation of Porous (Poly(lactide-co-glycolide)-co-(ε-caprolactone)) Polyurethane for Use in Orthopedic Scaffolds.

To develop an orthopedic scaffold that could overcome the limitations of implants used in clinics, we designed poly(ester-urethane) foams and compared their properties with those of a commercial gold standard. A degradable poly(ester-urethane) was synthetized by polyaddition between a diisocyanate poly(ε-caprolactone) prepolymer (PCL di-NCO, Mn = 2400 g·mol-1) and poly(lactic-co-glycolic acid) diol (PLGA, Mn = 2200 g·mol-1) acting as a chain extender. The resulting high-molecular-weight poly(ester-urethane) (PEU, Mn = 87,000 g·mol-1) was obtained and thoroughly characterized by NMR, FTIR and SEC-MALS. The porous scaffolds were then processed using the solvent casting (SC)/particle leaching (PL) method with different NaCl crystal concentrations. The morphology, pore size and porosity of the foams were evaluated using SEM, showing interconnected pores with a uniform size of around 150 µm. The mechanical properties of the scaffolds are close to those of the human meniscus (Ey = 0.5~1 MPa). Their degradation under accelerated conditions confirms that incorporating PLGA into the scaffolds greatly accelerates their degradation rate compared to the gold-standard implant. Finally, a cytotoxicity study confirmed the absence of the cytotoxicity of the PEU, with a 90% viability of the L929 cells. These results suggest that degradable porous PLGA/PCL poly(ester-urethane) has potential in the development of meniscal implants.

[Design and performance study of bone trabecular scaffolds based on triply periodic minimal surface method].

Triply periodic minimal surface (TPMS) is widely used because it can be used to control the shape of porous scaffolds precisely by formula. In this paper, an I-wrapped package (I-WP) type porous scaffolds were constructed. The finite element method was used to study the relationship between the wall thickness and period, the morphology and mechanical properties of the scaffolds, as well as to study the compression and fluid properties. It was found that the porosity of I-WP type scaffolds with different wall thicknesses (0.1 ~ 0.2 mm) and periods (I-WP 1 ~ I-WP 5) ranged from 68.01% ~ 96.48%, and the equivalent elastic modulus ranged from 0.655 ~ 18.602 GPa; the stress distribution of the scaffolds tended to be uniform with the increase of periods and wall thicknesses; the equivalent elastic modulus of the I-WP type scaffolds was basically unchanged after the topology optimization, and the permeability was improved by 52.3%. In conclusion, for the I-WP type scaffolds, the period parameter can be adjusted first, then the wall thickness parameter can be controlled. Topology optimization can be combined to meet the design requirements. The I-WP scaffolds constructed in this paper have good mechanical properties and meet the requirements of repairing human bone tissue, which may provide a new choice for the design of artificial bone trabecular scaffolds.

Effect of Internal Mechanical Environment of Porous Scaffolds on Mechano-driven Bone Ingrowth: A Numerical Study.

Porous cages with lower global stiffness induce more bone ingrowth and enhance bone-implant anchorage. However, it's dangerous for spinal fusion cages, which usually act as stabilizers, to sacrifice global stiffness for bone ingrowth. Intentional design on internal mechanical environment might be a promising approach to promote osseointegration without undermining global stiffness excessively. In this study, three porous cages with different architectures were designed to provide distinct internal mechanical environments for bone remodeling during spinal fusion process. A design space optimization-topology optimization based algorithm was utilized to numerically reproduce the mechano-driven bone ingrowth process under three daily load cases, and the fusion outcomes were analyzed in terms of bone morphological parameters and bone-cage stability. Simulation results show that the uniform cage with higher compliance induces deeper bone ingrowth than the optimized graded cage. Whereas, the optimized graded cage with the lowest compliance exhibits the lowest stress at the bone-cage interface and better mechanical stability. Combining the advantages of both, the strain-enhanced cage with locally weakened struts offers extra mechanical stimulus while keeping relatively low compliance, leading to more bone formation and the best mechanical stability. Thus, the internal mechanical environment can be well-designed via tailoring architectures to promote bone ingrowth and achieve a long-term bone-scaffold stability.

In silico and in vivo studies of the effect of surface curvature on the osteoconduction of porous scaffolds.

Recent evidence shows that the curvature of porous scaffold plays a significant role in guiding tissue regeneration. However, the underlying mechanism remains controversial to date. In this study, we developed an in silico model to simulate the effect of surface curvature on the osteoconduction of scaffold implants, which comprises the primary aspects of bone regeneration. Selective laser melting was used to manufacture a titanium scaffold with channels representative of different strut curvatures for in vivo assessment. The titanium scaffold was implanted in the femur condyles of rabbits to validate the mathematical model. Simulation results suggest that the curvature affected the distribution of growth factors and subsequently induced the migration of osteoblast lineage cells and bone deposition to the locations with higher curvature. The predictions of the mathematical model are in good agreement with the in vivo assessment results, in which newly formed bone first appeared adjacent to the vertices of the major axes in elliptical channels. The mechanism of curvature-guided osteoconduction may provide a guide for the design optimization of scaffold implants to achieve enhanced bone ingrowth.

High Molecular Weight Poly(glutamic acid) to Improve BMP2-Induced Osteogenic Differentiation.

FDA-approved bone morphogenetic protein 2 (BMP2) has serious side effects due to the super high dose requirement. Heparin is one of the most well-studied sulfated polymers to stabilize BMP2 and improve its functionality. However, the clinical use of heparin is questionable because of its undesired anticoagulant activity. Recent studies suggest that poly(glutamic acid) (pGlu) has the potential to improve BMP2 bioactivity with less safety concerns; however, the knowledge on pGlu's contribution remains largely unknown. Therefore, we aimed to study the role of pGlu in BMP2-induced osteogenesis and its potential application in bone tissue engineering. Our data, for the first time, indicated that both low (L-pGlu) and high molecular weight pGlu (H-pGlu) were able to significantly improve the BMP2-induced early osteoblastic differentiation marker (ALP) in MC3T3-E1 preosteoblasts. Importantly, the matrix mineralization was more rapidly enhanced by H-pGlu compared to L-pGlu. Additionally, our data indicated that only α-H-pGlu could significantly improve BMP2's activity, whereas γ-H-pGlu failed to do so. Moreover, both gene expression and mineralization data demonstrated that α-H-pGlu enabled a single dose of BMP2 to induce a high level of osteoblastic differentiation without multiple doses of BMP2. To study the potential application of pGlu in tissue engineering, we incorporated the H-pGlu+BMP2 nanocomplexes into the collagen hydrogel with significantly elevated osteoblastic differentiation. Furthermore, H-pGlu-coated 3D porous gelatin and chitosan scaffolds significantly enhanced osteogenic differentiation through enabling sustained release of BMP2. Thus, our findings suggest that H-pGlu is a promising new alternative with great potential for bone tissue engineering applications.

Improved cell seeding efficiency and cell distribution in porous hydroxyapatite scaffolds by semi-dynamic method.

Tissue engineering is a promising technique for the repair of bone defects. An efficient and homogeneous distribution of cell seeding into scaffold is a crucial but challenging step in the technique. Murine bone marrow mesenchymal stem cells were seeded into porous hydroxyapatite scaffolds of two morphologies by three methods: static seeding, semi-dynamic seeding, or dynamic perfusion seeding. Seeding efficiency, survival, distribution, and proliferation were quantitatively evaluated. To investigate the performance of the three seeding methods for larger/thicker scaffolds as well as batch seeding of numerous scaffolds, three scaffolds were stacked to form assemblies, and seeding efficiencies and cell distribution were analyzed. The semi-dynamic seeding and static seeding methods produced significantly higher seeding efficiencies, vitalities, and proliferation than did the dynamic perfusion seeding. On the other hand, the semi-dynamic seeding and dynamic perfusion seeding methods resulted in more homogeneous cell distribution than did the static seeding. For stacked scaffold assemblies, the semi-dynamic seeding method also created superior seeding efficiency and longitudinal cell distribution homogeneity. The semi-dynamic seeding method combines the high seeding efficiency of static seeding and satisfactory distribution homogeneity of dynamic seeding while circumventing their disadvantages. It may contribute to improved outcomes of bone tissue engineering.

Effect of Hydroxyapatite Coating by Er: YAG Pulsed Laser Deposition on the Bone Formation Efficacy by Polycaprolactone Porous Scaffold.

Composite scaffolds obtained by the combination of biodegradable porous scaffolds and hydroxyapatite with bone regeneration potential are feasible materials for bone tissue engineering. However, most composite scaffolds have been fabricated by complicated procedures or under thermally harsh conditions. We have previously demonstrated that hydroxyapatite coating onto various substrates under a thermally mild condition was achieved by erbium-doped yttrium aluminum garnet (Er: YAG) pulsed laser deposition (PLD). The purpose of this study was to prepare a polycaprolactone (PCL) porous scaffold coated with the hydroxyapatite by the Er: YAG-PLD method. Hydroxyapatite coating by the Er: YAG-PLD method was confirmed by morphology, crystallographic analysis, and surface chemical characterization studies. When cultured on PCL porous scaffold coated with hydroxyapatite, rat bone marrow-derived mesenchymal stem cells adhered, spread, and proliferated well. The micro-CT and staining analyses after the implantation of scaffold into the critical-sized calvaria bone defect in rats indicate that PCL porous scaffold coated with hydroxyapatite demonstrates accelerated and widespread bone formation. In conclusion, PCL porous scaffold coated with hydroxyapatite obtained by the Er: YAG-PLD method is a promising material in bone tissue engineering.

Application of an ultrasound semi-quantitative assessment in the degradation of silk fibroin scaffolds in vivo.

BACKGROUND: Research on the degradation of silk fibroin (SF) scaffolds in vivo lacks uniform and effective standards and experimental evaluation methods. This study aims to evaluate the application of ultrasound in assessing the degradation of SF scaffolds. METHODS: Two groups of three-dimensional regenerated SF scaffolds (3D RSFs) were implanted subcutaneously into the backs of Sprague-Dawley rats. B-mode ultrasound and hematoxylin and eosin (HE) staining were performed on days 3, 7, 14, 28, 56, 84, 112, 140, and 196. The cross-sectional areas for two groups of 3D RSFs that were obtained using these methods were semi-quantitatively analyzed and compared to evaluate the biodegradation of the implanted RSFs. RESULTS: The 3D RSFs in the SF-A group were wholly degraded at the 28th week after implantation. In contrast, the 3D RSFs in the SF-B group were completely degraded at the 16th week. Ultrasonic examination showed that the echoes of 3D RSFs in both groups gradually decreased with the increase of the implantation time. In the early stages of degradation, the echoes of the samples were higher than the echo of the muscle. In the middle of degeneration, the echoes were equal to the echo of the muscle. In the later stage, the echoes of the samples were lower than that of the muscle. The above changes in the SF-B group were earlier than those in the SF-A group. Semi-quantitative analysis of the cross-sectional areas detected using B-mode ultrasound revealed that the degradations of the two 3D RSF groups were significantly different. The degradation rate of the SF-B group was found to be higher than that of the SF-A group. This was consistent with the semi-quantitative detection results for HE staining. Regression analysis showed that the results of the B-mode ultrasound and HE staining were correlated in both groups, indicating that B-mode ultrasound is a reliable method to evaluate the SF scaffold degradation in vivo. CONCLUSIONS: This study suggests that B-mode ultrasound can clearly display the implanted SF scaffolds non-invasively and monitor the degradation of the different SF scaffolds after implantation in living organisms in real-time.

Mutual influence of selenium nanoparticles and FGF2-STAB® on biocompatible properties of collagen/chitosan 3D scaffolds: in vitro and ex ovo evaluation.

In a biological system, nanoparticles (NPs) may interact with biomolecules. Specifically, the adsorption of proteins on the nanoparticle surface may influence both the nanoparticles' and proteins' overall bio-reactivity. Nevertheless, our knowledge of the biocompatibility and risk of exposure to nanomaterials is limited. Here, in vitro and ex ovo biocompatibility of naturally based crosslinked freeze-dried 3D porous collagen/chitosan scaffolds, modified with thermostable fibroblast growth factor 2 (FGF2-STAB®), to enhance healing and selenium nanoparticles (SeNPs) to provide antibacterial activity, were evaluated. Biocompatibility and cytotoxicity were tested in vitro using normal human dermal fibroblasts (NHDF) with scaffolds and SeNPs and FGF2-STAB® solutions. Metabolic activity assays indicated an antagonistic effect of SeNPs and FGF2-STAB® at high concentrations of SeNPs. The half-maximal inhibitory concentration (IC50) of SeNPs for NHDF was 18.9 µg/ml and IC80 was 5.6 µg/ml. The angiogenic properties of the scaffolds were monitored ex ovo using a chick chorioallantoic membrane (CAM) assay and the cytotoxicity of SeNPs over IC80 value was confirmed. Furthermore, the positive effect of FGF2-STAB® at very low concentrations (0.01 µg/ml) on NHDF metabolic activity was observed. Based on detailed in vitro testing, the optimal concentrations of additives in the scaffolds were determined, specifically 1 µg/ml of FGF2-STAB® and 1 µg/ml of SeNPs. The scaffolds were further subjected to antimicrobial tests, where an increase in selenium concentration in the collagen/chitosan scaffolds increased the antibacterial activity. This work highlights the antimicrobial ability and biocompatibility of newly developed crosslinked collagen/chitosan scaffolds involving FGF2-STAB® and SeNPs. Moreover, we suggest that these sponges could be used as scaffolds for growing cells in systems with low mechanical loading in tissue engineering, especially in dermis replacement, where neovascularization is a crucial parameter for successful skin regeneration. Due to their antimicrobial properties, these scaffolds are also highly promising for tissue replacement requiring the prevention of infection.

Tuning Physicochemical Properties of a Macroporous Polysaccharide-Based Scaffold for 3D Neuronal Culture.

Central nervous system (CNS) lesions are a leading cause of death and disability worldwide. Three-dimensional neural cultures in biomaterials offer more physiologically relevant models for disease studies, toxicity screenings or in vivo transplantations. Herein, we describe the development and use of pullulan/dextran polysaccharide-based scaffolds for 3D neuronal culture. We first assessed scaffolding properties upon variation of the concentration (1%, 1.5%, 3% w/w) of the cross-linking agent, sodium trimetaphosphate (STMP). The lower STMP concentration (1%) allowed us to generate scaffolds with higher porosity (59.9 ± 4.6%), faster degradation rate (5.11 ± 0.14 mg/min) and lower elastic modulus (384 ± 26 Pa) compared with 3% STMP scaffolds (47 ± 2.1%, 1.39 ± 0.03 mg/min, 916 ± 44 Pa, respectively). Using primary cultures of embryonic neurons from PGKCre, Rosa26tdTomato embryos, we observed that in 3D culture, embryonic neurons remained in aggregates within the scaffolds and did not attach, spread or differentiate. To enhance neuronal adhesion and neurite outgrowth, we then functionalized the 1% STMP scaffolds with laminin. We found that treatment of the scaffold with a 100 µg/mL solution of laminin, combined with a subsequent freeze-drying step, created a laminin mesh network that significantly enhanced embryonic neuron adhesion, neurite outgrowth and survival. Such scaffold therefore constitutes a promising neuron-compatible and biodegradable biomaterial.

Multidimensional Mechanics of Three-Dimensional Printed and Micro-Architectured Scaffolds.

Mechanical properties of porous materials depend on their micro-architectural characteristics. Freeze casting is an effective method to fabricate micro-architectured porous scaffolds. Three key characteristics generated during freeze casting are wall thickness, number of domains at the cross-section, and transverse bridges connecting adjacent walls. To specifically study the effect of these structural characteristics on the mechanics and anisotropic compressive properties of scaffolds, we utilize additive manufacturing, i.e., 3D printing, to fabricate strictly designed cubic scaffolds with varying one characteristic at a time. We then compare strength, toughness, resilience, stiffness, and strain to failure in three orthogonal directions of the scaffolds, including longitudinal and transverse directions. To compare these multidimensional mechanics in a single diagram, we use a previously developed radar chart method to evaluate different scaffolds and unravel the effect of the structural characteristics. We find that the multidimensional mechanics can be effectively tuned by the micro-architectural characteristics. Notably, the buckling resistance of the scaffolds depends on all three structural characteristics. Our results show that an increased number of domains leads to enhanced toughness in all three directions. Increasing wall thickness leads to enhanced mechanical properties but comes at the price of losing small-sized pores, which is not favored for certain applications. In addition, adding transverse bridges increase not only the transverse strength of the scaffolds but also the longitudinal strength as they also enhance the buckling resistance. Our study provides important insights into the structure-property relationships of 3D-printed micro-architectured porous scaffolds.

Intra-aneurysmal embolization of cellulose porous beads to regenerate vessel wall: an experimental study.

PURPOSE: Although the treatment of intracranial cerebral aneurysms with detachable coils is now widely accepted, the problem of coil compaction and recanalization remains unsolved. If the vessel wall can be regenerated at the neck orifice of an aneurysm, thereby reducing the blood flow into the aneurysm, the recurrence rate of the aneurysm would decrease. Accordingly, we aimed to insert cellulose porous beads (CPBs) into rat models of external carotid artery (ECA) aneurysm and study their efficacy in promoting vessel wall regeneration. METHODS: Using a rat aneurysm model, we examined the tissue response to CPBs that were inserted into the ligated ECA sac of rats. The sacs were removed on days 14, 42, 84, and 180 after insertion and subjected to conventional and immunohistochemical examination. We evaluated the tissue response in the ECA sacs and observed the vessel wall regeneration progress. RESULTS: At the neck orifice of the aneurysm in which the CPB was inserted, a layer of regenerating α-smooth muscle actin-positive spindle cells was observed on day 14. The regenerative cell layer gradually thickened until day 42 and, thereafter, the thickness remained unchanged until day 180. A monolayer of factor VIII-positive cells also appeared at the neck orifice on day 14 and covered the entire orifice until day 180. The CPBs were stably localized in the sac without degradation or signs of inflammation. CONCLUSION: CPBs may be promising as embolic materials that can induce stable vessel wall regeneration at the neck orifice of an aneurysm without surrounding inflammatory reactions.

Evaluation of Proliferation and Osteogenic Differentiation of Human Umbilical Cord-Derived Mesenchymal Stem Cells in Porous Scaffolds.

INTRODUCTION: Human umbilical cord-derived mesenchymal stem cells (UCMSCs) are multiple potential stem cells that can differentiate into various kinds of functional cells, including adipocytes, osteoblasts, and chondroblasts. Thus, UCMSCs have recently been used in both stem cell therapy and tissue engineering applications to produce various functional tissues. This study aimed to evaluate the proliferation and differentiation of UCMSCs on porous scaffolds. METHODS: UCMSCs were established in a previous study and kept in liquid nitrogen. They were thawed and expanded in vitro to yield enough cells for further experiments. The cells were characterized as having MSC phenotype. They were seeded onto culture medium-treated porous scaffolds or on non-treated porous scaffolds at different densities of UCMSCs (105, 2.1 × 105, and 5 × 105 cells/0.005 g scaffold). The existence of UCMSCs on the scaffold was evaluated by nucleic staining using Hoechst 33342 dye, while cell proliferation on the scaffold was determined by MTT assay. Osteogenic differentiation was evaluated by changes in cellular morphology, accumulation of extracellular calcium, and expression of osteoblast-specific genes (including runx2, osteopontin (OPN), and osteocalcin (OCN)). RESULTS: The data showed that UCMSCs could attach, proliferate, and differentiate on both treated and non-treated scaffolds but were better on the treated scaffold. At a cell density of 105 cells/0.005 g scaffold, the adherent and proliferative abilities of UCMSCs were higher than that of the other densities after 14 days of culture (p < 0.05). Adherent UCMSCs on the scaffold could be induced into osteoblasts in the osteogenic medium after 21 days of induction. These cells accumulated calcium in the extracellular matrix that was positive with Alizarin Red staining. They also expressed some genes related to osteoblasts, including runx2, OPN, and OCN. CONCLUSION: UCMSCs could adhere, proliferate, and differentiate into osteoblasts on porous scaffolds. Therefore, porous scaffolds (such as Variotis) may be suitable scaffolds for producing bone tissue in combination with UCMSCs.

Enrichment of glioma stem cell-like cells on 3D porous scaffolds composed of different extracellular matrix.

Cancer stem cells (CSCs), being tumor-initiating with self-renewal capacity and heterogeneity, are most likely the cause of tumor resistance, reoccurrence and metastasis. To further investigate the role of CSCs in tumor biology, there is a need to develop an effective culture system to grow, maintain and enrich CSCs. Three-dimensional (3D) cell culture model has been widely used in tumor research and drug screening. Recently, researchers have begun to utilize 3D models to culture cancer cells for CSCs enrichment. In this study, glioma cell line was cultured with 3D porous chitosan (CS) scaffolds or chitosan-hyaluronic acid (CS-HA) scaffolds to explore the possibility of glioma stem cells (GSCs)-like cells enrichment, to study the morphology, gene expression, and in vivo tumorigenicity of 3D scaffolds cells, and to compare results to 2D controls. Results showed that glioma cells on both CS and CS-HA scaffolds could form tumor cell spheroids and increased the expression of GSCs biomarkers compared to conventional 2D monolayers. Furthermore, cells in CS-HA scaffolds had higher expression levels of epithelial-to-mesenchymal transition (EMT)-related gene. Specifically, the in vivo tumorigenicity capability of CS-HA scaffold cultured cells was greater than 2D cells or CS scaffold cultured cells. It is indicated that the chemical composition of scaffold plays an important role in the enrichment of CSCs. Our results suggest that CS-HA scaffolds have a better capability to enrich GSCs-like cells and can serve as a simple and effective way to cultivate and enrich CSCs in vitro to support the study of CSCs biology and development of novel anti-cancer therapies.

Porous Nanocomposite Comprising Ultralong Hydroxyapatite Nanowires Decorated with Zinc-Containing Nanoparticles and Chitosan: Synthesis and Application in Bone Defect Repair.

Hydroxyapatite nanowires exhibit a great potential in biomedical applications owing to their high specific surface area, high flexibility, excellent mechanical properties, and similarity to mineralized collagen fibrils of natural bone. In this work, zinc-containing nanoparticle-decorated ultralong hydroxyapatite nanowires (Zn-UHANWs) with a hierarchical nanostructure have been synthesized by a one-step solvothermal method. The highly flexible Zn-UHANWs exhibit a hierarchical rough surface and enhanced specific surface area as compared with ultralong hydroxyapatite nanowires (UHANWs). To evaluate the potential application of Zn-UHANWs in bone regeneration, the biomimetic Zn-UHANWs/chitosan (CS) (Zn-UHANWs/CS) composite porous scaffold with 80 wt % Zn-UHANWs was prepared by incorporating Zn-UHANWs into the chitosan matrix by the freeze-drying process. The as-prepared Zn-UHANWs/CS composite porous scaffold exhibits enhanced mechanical properties, highly porous structure, and excellent water retention capacity. In addition, the Zn-UHANWs/CS porous scaffold has a good biodegradability with the sustainable release of Zn, Ca, and P elements in aqueous solution. More importantly, the Zn-UHANWs/CS porous scaffold can promote the osteogenic differentiation of rat bone marrow derived mesenchymal stem cells and facilitate in vivo bone regeneration as compared with the pure CS porous scaffold or UHANWs/CS porous scaffold. Thus, both the Zn-UHANWs and Zn-UHANWs/CS porous scaffold developed in this work are promising for application in bone defect repair.

Construction of scaffolds composed of acellular cardiac extracellular matrix for myocardial tissue engineering.

High rates of mortality and morbidity stemming from cardiovascular diseases unveil extreme limitations in current therapies despite enormous advances in medical and pharmaceutical sciences. Following myocardial infarction (MI), parts of myocardium undergo irreversible remodeling and is substituted by a scar tissue which eventually leads to heart failure (HF). To address this issue, cardiac patches have been utilized to initiate myocardial regeneration. In this study, a porous cardiac patch is fabricated using a mixture of decellularized myocardium extracellular matrix (ECM) and chitosan (CS). Results of rheological measurements, SEM, biodegradation test, and MTT assay showed that the scaffold composed of 3.5% (w/w) CS and 0.5% ECM has the best potential in providing cardiac progenitor cells (CPCs) with a suitable microenvironmental condition for both attachment and growth. This study demonstrates that the fabricated scaffold is capable of transmitting both mechanical and chemical cues that is native to myocardial tissue and supports efficient growth of the CPCs.

Porous Scaffolds for Regeneration of Cartilage, Bone and Osteochondral Tissue.

Porous scaffolds play an important role as a temporary support for accommodation of seeded cells to control their functions and guide regeneration of functional tissues and organs. Various scaffolds have been prepared from biodegradable polymers and calcium phosphate. They have also been hybridized with bioactive factors to control differentiation of stem cells. Except the composition, porous structures of scaffolds are also extremely important for cell adhesion, spatial distribution and tissue regeneration. The method using preprepared ice particulates has been developed to precisely control surface and bulk pore structures of porous scaffolds. This chapter summarizes the design and preparation of porous scaffolds of biodegradable polymers and their hybrid scaffolds with calcium phosphate nanoparticles and bioactive factors. Their applications for regeneration of cartilage, bone and osteochondral tissue will be highlighted. HIGHLIGHTS: Porous scaffolds of naturally derived polymers and their hybrid scaffolds with biodegradable synthetic polymers have been prepared for cartilage tissue engineering. The surface and bulk pore structures of the scaffolds are controlled by using preprepared ice particulates. The scaffolds facilitate cartilage tissue engineering when they are used for three-dimension culture of chondrocytes. PLGA-collagen-BMP4 and collagen-CaP nanoparticles-dexamethasone hybrid scaffolds have been prepared and used for culture of mesenchymal stem cells. The hybrid scaffolds facilitate osteogenic differentiation of mesenchymal stem cells and ectopic bone tissue regeneration during in vitro culture and in vivo implantation. Osteochondral tissue engineering has been realized by laminating two different layers of cartilage and subchondral bone or by using stratified scaffolds for simultaneous regeneration of cartilage and subchondral bone.

Metabolic Oscillations in Co-Cultures of Hepatocytes and Mesenchymal Stem Cells: Effects of Seeding Arrangement and Culture Mixing.

In vitro assembly of functional liver tissue is a prerequisite for the transplantation of tissue-engineered livers. There is an increasing demand for in vitro models that replicate complex events occurring in the liver. However, tissue engineering of implantable liver systems is currently limited by the difficulty of assembling three dimensional hepatocyte cultures of a useful size, while maintaining full cell viability. Recent reports have demonstrated that bone marrow mesenchymal stem cells (BM-MSCs) can provide a number of cues promoting hepatocyte growth and development. In this study, the effects of BM-MSCs co-culture on hepatocyte metabolism were evaluated as a function of scaffold seeding arrangement. BM-MSCs were co-cultured with hepatocytes in porous chitosan-heparin scaffolds using several seeding arrangements. The seeded scaffolds were subjected to orbital shaking to enhance mass transfer. BM-MSC-hepatocyte co-cultures exhibited higher rates of hepatocyte-specific functions, compared to hepatocyte-only cultures, regardless of the seeding arrangement. Cells formed smaller-compact spheroids in the heterotypic systems compared to mono-cultures of hepatocytes only. The spheroids exhibited reduction in size with time in all conditions except for the condition where BM-MSCs were seeded one day after seeding hepatocytes. In this condition, spheroids increased in size due to BM-MSC proliferation. Spheroid size reduction was hypothesized to be the result of cyclic shear stresses generated by the orbital shaking. Furthermore, results suggested that BM-MSC seeding onto preformed hepatocyte spheroids provide a degree of shear-protection and trophic stimuli. Overall, the results indicate that co-culturing hepatocytes with BM-MSCs enhanced their metabolic functions for the first week of culture. J. Cell. Biochem. 118: 3003-3015, 2017. © 2017 Wiley Periodicals, Inc.

Modifications of collagen-based biomaterials with immobilized growth factors or peptides.

In order to provide an instructive microenvironment to facilitate cellular behaviors and tissue regeneration, biomaterials can be modified by immobilizing growth factors or peptides. We describe here our procedure for modification of collagen-based biomaterials, both porous scaffolds and hydrogel systems, with growth factors or peptides by covalent immobilization. Characterizations of the modified biomaterials (immobilization efficiency, release profile, morphology, mechanical strength, and rheology) and in vitro testing with cells are also discussed.

A mathematical model and computational framework for three-dimensional chondrocyte cell growth in a porous tissue scaffold placed inside a bi-directional flow perfusion bioreactor.

The in vitro chondrocyte cell culture for cartilage tissue regeneration in a perfusion bioreactor is a complex process. Mathematical modeling and computational simulation can provide important insights into the culture process, which would be helpful for selecting culture conditions to improve the quality of the developed tissue constructs. However, simulation of the cell culture process is a challenging task due to the complicated interaction between the cells and local fluid flow and nutrient transport inside the complex porous scaffolds. In this study, a mathematical model and computational framework has been developed to simulate the three-dimensional (3D) cell growth in a porous scaffold placed inside a bi-directional flow perfusion bioreactor. The model was developed by taking into account the two-way coupling between the cell growth and local flow field and associated glucose concentration, and then used to perform a resolved-scale simulation based on the lattice Boltzmann method (LBM). The simulation predicts the local shear stress, glucose concentration, and 3D cell growth inside the porous scaffold for a period of 30 days of cell culture. The predicted cell growth rate was in good overall agreement with the experimental results available in the literature. This study demonstrates that the bi-directional flow perfusion culture system can enhance the homogeneity of the cell growth inside the scaffold. The model and computational framework developed is capable of providing significant insight into the culture process, thus providing a powerful tool for the design and optimization of the cell culture process.