Caihong Zhu

Cellular modulation by the elasticity of biomaterials

The behaviors and functions of individual cells, fundamental to the complexity of multicellular organisms, are regulated by their integrated response to a variety of environmental cues such as soluble factors, extracellular matrix (ECM)-mediated signals, and cell-cell interactions. Among these cues, the biomechanical feature of the ECM, represented by its elasticity, has been increasingly recognized as a dominating factor of cell fate. This review article aims to provide an overview of the general principles and recent advances in the field of matrix elasticity-dependent regulation of cellular activities and functions, the underlying biomechanical and molecular mechanisms, as well as pathophysiological implications. A discussion is also provided as to how material design strategies can be used to control the local microenvironment of stem cells to direct their lineage commitment and functions toward tissue development and regeneration.

The effect of the fibre orientation of electrospun scaffolds on the matrix production of rabbit annulus fibrosus-derived stem cells.

Annulus fibrosus (AF) tissue engineering has recently received increasing attention as a treatment for intervertebral disc (IVD) degeneration; however, such engineering remains challenging because of the remarkable complexity of AF tissue. In order to engineer a functional AF replacement, the fabrication of cell-scaffold constructs that mimic the cellular, biochemical and structural features of native AF tissue is critical. In this study, we fabricated aligned fibrous polyurethane scaffolds using an electrospinning technique and used them for culturing AF-derived stem/progenitor cells (AFSCs). Random fibrous scaffolds, also prepared via electrospinning, were used as a control. We compared the morphology, proliferation, gene expression and matrix production of AFSCs on aligned scaffolds and random scaffolds. There was no apparent difference in the attachment or proliferation of cells cultured on aligned scaffolds and random scaffolds. However, compared to cells on random scaffolds, the AFSCs on aligned scaffolds were more elongated and better aligned, and they exhibited higher gene expression and matrix production of collagen-I and aggrecan. The gene expression and protein production of collagen-II did not appear to differ between the two groups. Together, these findings indicate that aligned fibrous scaffolds may provide a favourable microenvironment for the differentiation of AFSCs into cells similar to outer AF cells, which predominantly produce collagen-I matrix.

Modulation of the gene expression of annulus fibrosus-derived stem cells using poly(ether carbonate urethane)urea scaffolds of tunable elasticity.

UNLABELLED Annulus fibrosus (AF) injuries commonly lead to substantial deterioration of the intervertebral disc (IVD). While tissue engineering has recently evolved into a promising approach for AF regeneration, it remains challenging due to the cellular, biochemical, and mechanical heterogeneity of AF tissue. In this study, we explored the use of AF-derived stem cells (AFSCs) to achieve diversified differentiation of cells for AF tissue engineering. Since the differentiation of stem cells relies significantly on the elasticity of the substrate, we synthesized a series of biodegradable poly(ether carbonate urethane)urea (PECUU) materials whose elasticity approximated that of native AF tissue. When AFSCs were cultured on electrospun PECUU fibrous scaffolds, the gene expression of collagen-I in the cells increased with the elasticity of scaffold material, whereas the expression of collagen-II and aggrecan genes showed an opposite trend. At the protein level, the content of collagen-I gradually increased with substrate elasticity, while collagen-II and GAG contents decreased. In addition, the cell traction forces (CTFs) of AFSCs gradually decreased with scaffold elasticity. Such substrate elasticity-dependent changes of AFSCs were similar to the gradual transition in the genetic, biochemical, and biomechanical characteristics of cells from inner to outer regions of native AF tissue. Together, findings from this study indicate that AFSCs, depending on the substrate elasticity, have strong tendencies to differentiate into various types of AF-like cells, thereby providing a solid foundation for the tissue engineering applications of AFSCs. STATEMENT OF SIGNIFICANCE Repairing the annulus fibrosus (AF) of intervertebral disc (IVD) is critical for the treatment of disc degeneration disease, but remains challenging due to the significant heterogeneity of AF tissue. Previously, we have identified rabbit AF-derived stem cells (AFSCs), which are AF tissue-specific and hold promise for AF regeneration. In this study, we synthesized a series of poly(ether carbonate urethane)ureas of various elasticity (or stiffness) and explored the potential of induced differentiation of AFSCs using electrospun PECUU scaffolds. This work has, for the first time, found that AFSCs are able to present different gene expression patterns simply as a result of the elasticity of scaffold material. Therefore, our findings will help supplement current knowledge of AF tissue regeneration and may benefit a diversified readership from scientific, engineering, and clinical settings whose work involves the biology and tissue engineering of IVD.

Gene expression modulation in TGF-β3-mediated rabbit bone marrow stem cells using electrospun scaffolds of various stiffness

Tissue engineering has recently evolved into a promising approach for annulus fibrosus (AF) regeneration. However, selection of an ideal cell source, which can be readily differentiated into AF cells of various regions, remains challenging because of the heterogeneity of AF tissue. In this study, we set out to explore the feasibility of using transforming growth factor‐β3‐mediated bone marrow stem cells (tBMSCs) for AF tissue engineering. Since the differentiation of stem cells significantly relies on the stiffness of substrate, we fabricated nanofibrous scaffolds from a series of biodegradable poly(ether carbonate urethane)‐urea (PECUU) materials whose elastic modulus approximated that of native AF tissue. We cultured tBMSCs on PECUU scaffolds and compared their gene expression profile to AF‐derived stem cells (AFSCs), the newly identified AF tissue‐specific stem cells. As predicted, the expression of collagen‐I in both tBMSCs and AFSCs increased with scaffold stiffness, whereas the expression of collagen‐II and aggrecan genes showed an opposite trend. Interestingly, the expression of collagen‐I, collagen‐II and aggrecan genes in tBMSCs on PECUU scaffolds were consistently higher than those in AFSCs regardless of scaffold stiffness. In addition, the cell traction forces (CTFs) of both tBMSCs and AFSCs gradually decreased with scaffold stiffness, which is similar to the CTF change of cells from inner to outer regions of native AF tissue. Together, findings from this study indicate that tBMSCs had strong tendency to differentiate into various types of AF cells and presented gene expression profiles similar to AFSCs, thereby establishing a rationale for the use of tBMSCs in AF tissue engineering.

Microsol-electrospinning for controlled loading and release of water-soluble drugs in microfibrous membranes

Water-solubility facilitates drug transportation and distribution of drugs throughout the body and hence effectively promotes their absorption. While there have been a number of techniques for incorporating water-soluble drugs into electrospun fibers to realize sustained release of them, problems including burst and uncontrolled release still remain to be solved. In this study, we developed a microsol-electrospinning technique for fabricating core–shell microfibers to achieve incubated, controlled and sustainable release of water-soluble drugs such as chloroquine (CQ). In this approach, nanoparticles made of CQ-loaded hyaluronic acid (HA) sol were first prepared using the emulsification method. Next, the HA-sol nanoparticles were dispersed in poly(L-lactide) (PLLA) electrospinning solution to form a uniform suspension, which was used for fabricating composite microfibers through microsol-electrospinning. Judging from SEM and TEM, the composite microfibers had smooth, uniform morphology and core–shell structure. Further tests showed that the microsol-electrospun microfibers had similar physical, chemical, and mechanical properties as microfibers fabricated using a conventional electrospinning approach. In vitro drug release tests showed that compared to conventional electrospun microfibers, the burst release of CQ was significantly reduced in microsol-electrospun microfibers. Meanwhile, the release time of CQ was markedly extended, being as long as more than 40 days. Importantly, the drug release rate could be readily adjusted by changing the concentration of microsol particles and the amount of drug in the microfibers. Together, findings from this study have revealed that microsol-electrospinning is a facile technique for loading water-soluble drugs into electrospun microfibers and releasing them in a controlled fashion, which may expand the applications of water-soluble drugs.

Nanogel-electrospinning for controlling the release of water-soluble drugs

Application of electrospun fibers for the purpose of loading and controlled release of water-soluble drugs remains a challenge due to their low carrying effect as well as quick and unstable drug release. In this study, we have developed a novel nanogel-electrospinning technology through which more stable loading and prolonged release of water-soluble drugs was achieved. In brief, nanogel particles synthesized from a chloroquine (CQ)-loaded bovine serum albumin (BSA) solution were prepared and then combined with genipin, a crosslinking agent. The nanogel solution was then crosslinked to prepare an electrospinning solution with an inner mesh structure. Finally, the microfibrous membranes were fabricated by electrospinning the solution. Uniform BSA nanogel particles were wrapped in the fiber membrane and the number of particles increased with the increase of BSA and genipin concentrations. In addition to being loaded within the BSA nanogel particles, CQ was distributed in the fibers as well, which could be clearly identified using ultraviolet-visible spectroscopy (UV-Vis). The physical, chemical, and mechanical properties of nanogel-electrospun microfibers were similar to those of microfibers formed through a conventional electrospinning approach. The drug release tests indicated that with the same number of BSA nanogel particles, increased CQ loading resulted in increased initial release of the same. The duration of a single drug release cycle lasted up to 40 days. In conclusion, findings from this study have indicated that nanogel-electrospinning is a convenient and effective technology to achieve controlled long-term release of water-soluble drugs.

Effect of scaffold elasticity on the gene expression of annulus fibrosus-derived stem cells.

This article provides more experimental details and findings of the study as to how the elasticity of scaffold material modulates the gene expression of annulus fibrosus-derived stem cells (AFSCs) (Zhu et al., 2015 [1]). The detailed synthetic route and characterizations of four kinds of biodegradable poly(ether carbonate urethane)ureas (PECUUs) are described. After AFSCs were cultured on electrospun PECUU fibrous scaffolds, the cell proliferation and gene expression analyses were performed to explore the effect of substrate elasticity on the growth and differentiation characteristics of AFSCs.

In situ silk fibroin-mediated crystal formation of octacalcium phosphate and its application in bone repair

The development of an ideal scaffold material is critical for the repair of bone defects. Being an important precursor of the mineralized matrix of bone tissue, octacalcium phosphate (OCP) has been considered a promising bone substitute. However, its application is largely limited due to the thermodynamical instability and poor processability of it. In this study, OCP was prepared by co-precipitation in the presence of small amount of silk fibroin (SF), which regulated the crystallization of OCP and led to the formation of SF-OCP complex. The diameters of OCP crystals in OCP, 0.1SF-OCP, 0.3SF-OCP and 1SF-OCP complexes were 489.0 ± 399.1 nm, 102.2 ± 50.7 nm, 94.7 ± 48.4 nm and 223.7 ± 167.6 nm, respectively. However, the shape of OCP crystals did not apparently change by the presence of SF. Further, porous SF/OCP composite scaffolds with pore size of 111.9 ± 33.1 μm were prepared, in which small crystals of SF-OCP complex were embedded in a SF matrix. MC3T3-E1 cells could attach and proliferate well on both the rugged surfaces and the pores of SF/OCP scaffolds, indicating their decent biocompatibility. Further, SF/OCP scaffolds markedly promoted bone regeneration in a rat calvarial critical-sized defect model. Both micro-CT and H&E characterizations showed that bone formation not only occurred around the scaffolds, but also penetrated into their center. Therefore, such SF/OCP composite scaffolds may have potential applications in bone tissue engineering.

The “Magnesium Sacrifice” Strategy Enables PMMA Bone Cement Partial Biodegradability and Osseointegration Potential

Poly (methyl methacrylate) (PMMA)-based bone cements are the most commonly used injectable orthopedic materials due to their excellent injectability and mechanical properties. However, their poor biocompatibility and excessive stiffness may cause complications such as aseptic implant loosening and stress shielding. In this study, we aimed to develop a new type of partially biodegradable composite bone cement by incorporating magnesium (Mg) microspheres, known as “Mg sacrifices” (MgSs), in the PMMA matrix. Being sensitive to the physiological environment, the MgSs in PMMA could gradually degrade to produce bioactive Mg ions and, meanwhile, result in an interconnected macroporous structure within the cement matrix. The mechanical properties, solidification, and biocompatibility, both in vitro and in vivo, of PMMA–Mg bone cement were characterized. Interestingly, the incorporation of Mg microspheres did not markedly affect the mechanical strength of bone cement. However, the maximum temperature upon setting of bone cement decreased. This partially biodegradable composite bone cement showed good biocompatibility in vitro. In the in vivo study, considerable bony ingrowth occurred in the pores upon MgS degradation. Together, the findings from this study indicate that such partially biodegradable PMMA–Mg composite may be ideal bone cement for minimally invasive orthopedic surgeries such as vertebroplasty and kyphoplasty.