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The paracrine and autocrine processes of the host response play an integral role in the success of scaffold-based tissue regeneration. Recently, the immunomodulatory scaffolds have received huge attention for modulating inflammation around the host tissue through releasing anti-inflammatory cytokine. However, controlling the inflammation and providing a sustained release of anti-inflammatory cytokine from the scaffold in the digestive inflammatory environment are predicated upon a comprehensive understanding of three fundamental questions. (1) How does the release rate of cytokine from the scaffold change in the digestive inflammatory environment? (2) Can we prevent the premature scaffold degradation and burst release of the loaded cytokine in the digestive inflammatory environment? (3) How does the scaffold degradation prevention technique affect the immunomodulatory capacity of the scaffold? This study investigated the impacts of the digestive inflammatory environment on scaffold degradation and how pre-mature degradation can be prevented using genipin crosslinking and how genipin crosslinking affects the interleukin-4 (IL-4) release from the scaffold and differentiation of naïve macrophages (M0). Our results demonstrated that the digestive inflammatory environment (DIE) attenuates protein retention within the scaffold. Over 14 days, the encapsulated protein released 46% more in DIE than in phosphate buffer saline (PBS), which was improved through genipin crosslinking. We have identified the 0.5 ( w / v ) genipin concentration as an optimal concentration for improved IL-4 released from the scaffold, cell viability, mechanical strength, and scaffold porosity, and immunomodulation studies. The IL-4 released from the injectable scaffold could differentiate naïve macrophages to an anti-inflammatory (M2) lineage; however, upon genipin crosslinking, the immunomodulatory capacity of the scaffold diminished significantly, and pro-inflammatory markers were expressed dominantly.

Recurrence of intervertebral disc (IVD) herniation is the most important factor leading to chronic low back pain and subsequent disability after discectomy. Efficacious annulus fibrosus (AF) repair strategy that delivers cells and biologics to IVD injury site is needed to limit the progression of disc degeneration and promote disc self-regeneration capacities after discectomy procedures. In this study, a biphasic mechanically-conditioned scaffold encapsulated with human adipose-derived stem cells (ASCs) is studied as a potential treatment strategy for AF defects. Equiaxial strains and frequencies were applied to ASCs-encapsulated scaffolds to identify the optimal loading modality to induce AF differentiation. Equiaxial loading resulted in 2-4 folds increase in secretion of extracellular matrix proteins and the reorganization of the matrix fibers and elongations of the cells along the load direction. Further, the equiaxial load induced region-specific differentiation of ASCs within the inner and outer regions of the biphasic scaffolds. Gene expression of AF markers was upregulated with 5-30 folds within the equiaxially loaded biphasic scaffolds compared to unstrained samples. The results suggest that there is a specific value of equiaxial strain favorable to differentiate ASCs towards AF lineage and that ASCs-embedded biphasic scaffold can potentially be utilized to repair the AF defects.

Successful tissue engineering and regenerative therapy necessitate having extensive knowledge about mechanical milieu in engineered tissues and the resident cells. In this study, we have merged two powerful analysis tools, namely finite element analysis and stochastic analysis, to understand the mechanical strain within the tissue scaffold and residing cells and to predict the cell viability upon applying mechanical strains. A continuum-based multi-length scale finite element model (FEM) was created to simulate the physiologically relevant equiaxial strain exposure on cell-embedded tissue scaffold and to calculate strain transferred to the tissue scaffold (macro-scale) and residing cells (micro-scale) upon various equiaxial strains. The data from FEM were used to predict cell viability under various equiaxial strain magnitudes using stochastic damage criterion analysis. The model validation was conducted through mechanically straining the cardiomyocyte-encapsulated collagen constructs using a custom-built mechanical loading platform (EQUicycler). FEM quantified the strain gradients over the radial and longitudinal direction of the scaffolds and the cells residing in different areas of interest. With the use of the experimental viability data, stochastic damage criterion, and the average cellular strains obtained from multi-length scale models, cellular viability was predicted and successfully validated. This methodology can provide a great tool to characterize the mechanical stimulation of bioreactors used in tissue engineering applications in providing quantification of mechanical strain and predicting cellular viability variations due to applied mechanical strain.

It is crucial to replicate the micromechanical milieu of native tissues to achieve efficacious tissue engineering and regenerative therapy. In this study, we introduced an innovative loading platform, EQUicycler, that utilizes a simple, yet effective, and well-controlled mechanism to apply physiologically relevant homogenous mechanical equiaxial strain on three-dimensional cell-embedded tissue scaffolds. The design of EQUicycler ensured elimination of gripping effects through the use of biologically compatible silicone posts for direct transfer of the mechanical load to the scaffolds. Finite Element Modeling (FEM) was created to understand and to quantify how much applied global strain was transferred from the loading mechanism to the tissue constructs. In vitro studies were conducted on various cell lines associated with tissues exposed to equiaxial mechanical loading in their native environment. In vitro results demonstrated that EQUicycler was effective in maintaining and promoting the viability of different musculoskeletal cell lines and upregulating early differentiation of osteoprogenitor cells. By utilizing EQUicycler, collagen fibers of the constructs were actively remodeled. Residing cells within the collagen construct elongated and aligned with strain direction upon mechanical loading. EQUicycler can provide an efficient and cost-effective tool to conduct mechanistic studies for tissue engineered constructs designed for tissue systems under mechanical loading in vivo., Competing Interests: The authors declare that there are no competing interests regarding the publication of this paper.

In this work, we developed a nanofibrous, yet injectable orthobiologic tissue scaffold that is capable of hosting osteoprogenitor cells and controlling kinetic release profile of the encapsulated pro-osteogenic factor without diminishing its bioactivity over 21days. This innovative injectable scaffold was synthesized by incorporating electrospun and subsequently O2 plasma-functionalized polycaprolactone (PCL) nanofibers within the collagen type-I solution along with MC3T3-E1 cells (pre-osteoblasts) and bone morphogenetic protein-2 (BMP2). Through changing the PCL nanofiber concentration within the injectable scaffolds, we were able to tailor the mechanical strength, protein retention capacity, bioactivity preservation, and osteoinductive potential of the scaffolds. The nanofibrous internal structure of the scaffold allowed us to use a low dose of BMP2 (200ng/ml) to achieve osteoblastic differentiation in in vitro culture. The osteogenesis capacity of the injectable scaffolds were evaluated though measuring MC3T3-E1 cell proliferation, ALP activity, matrix mineralization, and early- and late-osteoblast specific gene expression profiles over 21days. The results demonstrated that the nanofibrous injectable scaffold provides not only an osteoinductive environment for osteoprogenitor cells to differentiate, but also a suitable biomechanical and biochemical environment to act as a reservoir for osteogenic factors with controlled release profile., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The objective of this study was to understand the temporal relationship between in situ generated calcium content (mineralization) and the mechanical properties of an injectable orthobiologic bone-filler material. Murine derived osteoblast progenitor cells were differentiated using osteogenic factors and encapsulated within an injectable polycaprolactone nanofiber-collagen composite scaffold (PN-COL +osteo) to evaluate the effect of mineralization on the mechanical properties of the PN-COL scaffold. A comprehensive study was conducted using both an experimental and a predictive analytical mechanical analysis for mechanical property assessment as well as an extensive in vitro biological analysis for in situ mineralization. Cell proliferation was evaluated using a PicoGreen dsDNA quantification assay and in situ mineralization was analyzed using both an alkaline phosphatase (ALP) assay and an Alizarin Red stain-based assay. Mineralized matrix formation was further evaluated using energy dispersive x-ray spectroscopy (EDS) and visualized using SEM and histological analyses. Compressive mechanical properties of the PN-COL scaffolds were determined using a confined compression stress-relaxation protocol and the obtained data was fit to the standard linear solid viscoelastic material mathematical model to demonstrate a relationship between increased in situ mineralization and the mechanical properties of the PN-COL scaffold. Cell proliferation was constant over the 21 day period. ALP activity and calcium concentration significantly increased at day 14 and 21 as compared to PN-COL -osteo with undifferentiated osteoblast progenitor cells. Furthermore, at day 21 EDS, SEM and von Kossa histological staining confirmed mineralized matrix formation within the PN-COL scaffolds. After 21 days, compressive modulus, peak stress, and equilibrium stress demonstrate significant increases of 3.4-fold, 3.3-fold, and 4.0-fold respectively due to in situ mineralization. Viscoelastic parameters calculated through the standard linear solid mathematical model fit to the stress-relaxation data also indicate improved mechanical properties after in situ mineralization. This investigation clearly demonstrates that in situ mineralization can increase the mechanical properties of an injectable orthobiologic scaffold and can possibly guide the design of an effective osteoconductive injectable material., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

There is an increasing demand for an injectable cell coupled three-dimensional (3D) scaffold to be used as bone fracture augmentation material. To address this demand, a novel injectable osteogenic scaffold called PN-COL was developed using cells, a natural polymer (collagen type-I), and a synthetic polymer (polycaprolactone (PCL)). The injectable nanofibrous PN-COL is created by interspersing PCL nanofibers within pre-osteoblast cell embedded collagen type-I. This simple yet novel and powerful approach provides a great benefit as an injectable bone scaffold over other non-living bone fracture stabilization polymers, such as polymethylmethacrylate and calcium content resin-based materials. The advantages of injectability and the biomimicry of collagen was coupled with the structural support of PCL nanofibers, to create cell encapsulated injectable 3D bone scaffolds with intricate porous internal architecture and high osteoconductivity. The effects of PCL nanofiber inclusion within the cell encapsulated collagen matrix has been evaluated for scaffold size retention and osteocompatibility, as well as for MC3T3-E1 cells osteogenic activity. The structural analysis of novel bioactive material proved that the material is chemically stable enough in an aqueous solution for an extended period of time without using crosslinking reagents, but it is also viscous enough to be injected through a syringe needle. Data from long-term in vitro proliferation and differentiation data suggests that novel PN-COL scaffolds promote the osteoblast proliferation, phenotype expression, and formation of mineralized matrix. This study demonstrates for the first time the feasibility of creating a structurally competent, injectable, cell embedded bone tissue scaffold. Furthermore, the results demonstrate the advantages of mimicking the hierarchical architecture of native bone with nano- and micro-size formation through introducing PCL nanofibers within macron-size collagen fibers and in promoting osteoblast phenotype progression for bone regeneration.