Tissue Engineering Part A: Research Advances

The current White paper summarizes the discussions and exchange of experiences during the first European Interdisciplinary Summit on Cell-Based ATMPs held in Vienna, Austria, May 02–03, 2013. The meeting was supported by the Research Networking Programme REMEDIC (regenerative medicine) funded by the European Science Foundation and by the British Medical Research Council. To improve the competitiveness of Europe in the field of cell-based Advanced Medicinal Therapy Products (ATMPs), the following key issues were identified during the meeting: removal of national hurdles in the European Union, harmonization of national and subnational differences in Hospital Exemption rules, improved treatment algorithms for reimbursement, better knowledge on the mode of action, predictive preclinical efficacy and safety testing, need for innovative systems for preclinical testing, appropriate product characterization, manufacturing with cost of goods in mind, and appropriate design of clinical trials.

Medical innovation occupies a position somewhere between standard practice and clinical research, but innovation is primarily intended to benefit an individual patient where standard treatment fails. Medical innovations in the area of regenerative medicine have the potential to completely transform medical practice, but rely upon some major revision to the nature of treatments beyond drug-based therapies. There is considerable investment in scientific and clinical research, but further attention could be paid to legal barriers to medical innovation imposed by the threat of medical malpractice. We survey in this article the legal framework for making determinations of medical malpractice in general, and highlight the issues specific to innovative treatments. In essence, liability could be imposed for failing to adequately inform the patient about the innovative nature of the suggested therapy or based on the fact that the risks outweighed the benefits. As for the latter, we examine whether liability is likely to be based merely on deviating from existing practice or on an examination on the merits of the treatments' risks and benefits. The facts that some risks are unforeseeable and some benefits are external to the patient complicate negligence determinations. The first fact relates to the problem of judging adverse events in hindsight; the second, to the obligation to make decisions based on the patient's best interest and avoid conflict of interests. In addition, we evaluate the relationship between the obligations to secure the patient's informed consent and to avoid clinical negligence. We identify the need for further research to examine the significance of the putative anti-innovation bias that current liability regimen has, and to examine whether a move to strict liability might avoid such bias, while being fair to patients who contribute for the advancement of medical knowledge by participating in innovative therapies.

Tissue engineering (TE) is a scientific field that will have an influence on our daily lives. It has the potential to revolutionize medical treatments, but it has also an impact on our human image and is associated with potential risks and ethical aspects. Among the publicly controversial issues are embryonic and induced pluripotent stem cells, cloning, uncertainties regarding risks and informed consent issues. To maintain public confidence in the science of TE, a good solution is public dialogues with patients and other interested lay people that gives the public the chance to independently evaluate TE issues and build their own opinion based on information from different perspectives. The article describes public participation projects in TE on stem cell research and gene therapy and presents the case study of the EU-Gene Activated Matrices for Bone and Cartilage Regeneration on Arthritis (GAMBA) panels, a dialogue with patient and citizen panels in three European countries. In the GAMBA panels, lay participants assessed the basic research project aimed at finding ways of healing osteoarthritis through a matrix composed of adult stem cells, gene vectors, nanoparticles, and biomaterials. The results of the dialogues in different countries, such as Denmark, Japan, Ireland, Switzerland, and Germany, are compared and the evaluation criteria for high quality dialogues are presented, including multiperspectivity, openness of results, a clear mandate, impartial facilitation of the panels, and transparency.

Stem cell-based interventions provide new treatment prospects for many disease conditions, including cardiovascular disorders. Clinical trials are necessary to collect adequate evidence on (long-term) safety and efficacy of novel interventions such as stem cells, but the design and launch of clinical trials, from first-in-human studies to larger randomized controlled trials (RCTs), is scientifically and ethically challenging. Stem cells are different from traditional pharmaceuticals, surgical procedures, and medical devices in the following ways: the novelty and complexity of stem cells, the invasiveness of the procedures, and the novel aim of regeneration. These specifics, combined with the characteristics of the study population, will have an impact on the design and ethics of RCTs. The recently closed JUVENTAS trial will serve as an example to identify the (interwoven) scientific and ethical challenges in the design and launch of stem cell RCTs. The JUVENTAS trial has investigated the efficacy of autologous bone marrow cells in end-stage vascular patients, in a double-blind sham-controlled design. We first describe the choices, considerations, and experiences of the JUVENTAS team. Subsequently, we identify the main ethical and scientific challenges and discuss what is important to consider in the design of future stem cell RCTs: assessment of risks and benefits, the choice for outcome measures, the choice for the comparator, the appropriate selection of participants, and adequate informed consent. Additionally, the stem cell field is highly in the spotlight due to the (commercial) interests and expectations. This warrants a cautious pace of translation and scrupulous set up of clinical trials, as failures could put the field in a negative light. At the same time, knowledge from clinical trials is necessary for the field to progress. We conclude that in the scientifically and ethically challenging field of stem cell RCTs, researchers and clinicians have to maneuver between the Skylla of hyper accelerated translation without rigorously conducted RCTs and the Charybdis of the missed opportunity of valuable knowledge.

Over the last 2 years a global assessment of stem cell engineering (SCE) was conducted with the sponsorship of the National Science Foundation, the National Cancer Institute at the National Institutes of Health, and the National Institute of Standards and Technology. The purpose was to gather information on the worldwide status and trends in SCE, that is, the involvement of engineers and engineering approaches in the stem cell field, both in basic research and in the translation of research into clinical applications and commercial products. The study was facilitated and managed by the World Technology Evaluation Center. The process involved site visits in both Asia and Europe, and it also included several different workshops. From this assessment, the panel concluded that there needs to be an increased role for engineers and the engineering approach. This will provide a foundation for the generation of new markets and future economic growth. To do this will require an increased investment in engineering, applied research, and commercialization as it relates to stem cell research and technology. It also will require programs that support interdisciplinary teams, new innovative mechanisms for academic–industry partnerships, and unique translational models. In addition, the global community would benefit from forming strategic partnerships between countries that can leverage existing and emerging strengths in different institutions. To implement such partnerships will require multinational grant programs with appropriate review mechanisms.

Akt kinase is a central signal transduction node that integrates extracellular cues that regulate cell migratory, proliferative, and morphological functions during angiogenesis. However, how Akt activity is modulated and contributes to subsequent vessel maturation is unclear. In this study we investigated the role of Akt1 in vessel maturation using human dermal microvascular endothelial cells (HDMVECs) expressing constitutively active and hemiphosphorylated Akt1 epi-alleles with graded kinase activity. HDMVECs expressing Akt1 epi-alleles were analyzed in vivo in a tissue engineering setting using a model of angiogenesis comprising cell-seeded poly-L-lactic acid scaffolds implanted subcutaneously into NOD/SCID murine hosts. The resultant intraimplant microvasculature was quantified for vascular parameters, including vessel diameter, perfusion, vascular density, and pericyte coverage. We found that constitutive Akt1 kinase activity in implanted HDMVECs correlated with loss of neovasculature function. Further, we found that the presence of coimplanted vascular smooth muscle cells (vSMCs) in the implants failed to promote blood vessel growth and maturation in a graded, Akt1 kinase activity-dependent manner. These results indicate that constitutive Akt1 activity disrupts the normal blood vessel growth and maturation. Therefore, we suggest that a downregulation of Akt1 activity is necessary for vSMC-induced maturation of newly formed blood vessels to occur.

The natural reparative mechanisms triggered by tendon damage often lead to the formation of biomechanically inferior scar tissue that is prone to re-injury. Before the efficient application of stem cell-based regenerative therapies, the processes regulating tenocyte differentiation should first be better understood. Three-dimensional (3D) growth environments under strain and the exogenous addition of transforming growth factor beta3 (TGF-β3) have separately been shown to promote tendon differentiation. The aim of this study was to determine the ability of both of these factors to induce tendon differentiation of equine embryo-derived stem cells (ESCs). ESCs seeded into 3D collagen constructs can contract the matrix to a similar degree to that of tenocyte-seeded constructs and histologically appear nearly identical, with no areas of cartilage or bone tissue deposition. Tendon-associated genes and proteins Tenascin-C, Collagen Type I, and COMP are significantly up-regulated in the 3D ESC constructs compared with tenogenic induction in monolayer ESC cultures. The addition of TGF-β3 to the 3D cultures further up-regulates the expression of these genes and also induces the expression of mature tenocyte markers Tenomodulin and Thrombospondin-4. Our results show that when ESCs are exposed to the intrinsic forces exerted by a 3D culture environment, they express tendon-associated genes and proteins which are indicative of tenocyte lineage differentiation and that this effect is synergistically enhanced and accelerated by the addition of TGF-β3.

On activation, platelets secrete an array of growth factors that contribute to bone regeneration. Combining platelet-rich plasma (PRP) with bone graft substitutes has the potential to reduce or replace the reliance on autografts. Lack of standardization and improper use may contribute to the conflicting outcomes reported within both preclinical and clinical investigations using PRP. This study investigates the effect of PRP dose on bone augmentation. Eighty critical-sized defects were created in the cancellous bone of the medial proximal tibia and the distal femur of 20 five-year-old female sheep. The defects were treated with three doses of an autologous thrombin-activated PRP combined with a biphasic calcium phosphate (BCP) or autograft and empty defects. Radiography, micro-computed tomography, histology, histomorphometry, and fluorochrome bone labels were examined at 4 weeks. The empty defects did not spontaneously heal. The highest dose of PRP treatment had a significantly greater micro-CT bone volume/total volume compared with the BCP alone (PRP: 30.6%±1.8%; BCP: 24.5%±0.1%). All doses of PRP treatment were significantly greater than the BCP alone for histomorphometric new bone area (PRP: 14.5%±1.3%; BCP: 9.7%±1.5%) and bone ingrowth depth (PRP: 2288±210 μm; BCP:1151±268 μm). From week 2 onward, PRP had a significant effect on the weekly bone ingrowth compared with BCP; however, autografts had the highest amount of weekly fluorescent bone labeling. PRP induces new bone formation with a dose-dependent response at 4 weeks when used with a BCP in sheep.

The recapitulation of the material properties and structure of the native aortic valve leaflet, specifically its anisotropy and laminate structure, is a major design goal for scaffolds for heart valve tissue engineering. Poly(ethylene glycol) (PEG) hydrogels are attractive scaffolds for this purpose as they are biocompatible, can be modified for their mechanical and biofunctional properties, and can be laminated. This study investigated augmenting PEG hydrogels with polycaprolactone (PCL) as an analog to the fibrosa to improve strength and introduce anisotropic mechanical behavior. However, due to its hydrophobicity, PCL must be modified prior to embedding within PEG hydrogels. In this study, PCL was electrospun (ePCL) and modified in three different ways, by protein adsorption (pPCL), alkali digestion (hPCL), and acrylation (aPCL). Modified PCL of all types maintained the anisotropic elastic moduli and yield strain of unmodified anisotropic ePCL. Composites of PEG and PCL (PPCs) maintained anisotropic elastic moduli, but aPCL and pPCL had isotropic yield strains. Overall, PPCs of all modifications had elastic moduli of 3.79±0.90 MPa and 0.46±0.21 MPa in the parallel and perpendicular directions, respectively. Valvular interstitial cells seeded atop anisotropic aPCL displayed an actin distribution aligned in the direction of the underlying fibers. The resulting scaffold combines the biocompatibility and tunable fabrication of PEG with the strength and anisotropy of ePCL to form a foundation for future engineered valve scaffolds.

Repairing articular cartilage by combining microfracture and various scaffolds has been extensively performed in in vivo animal models. We previously described a novel extracellular matrix (ECM) scaffold for cartilage tissue engineering. The aim of this study was to investigate the effect of a bone marrow-derived mesenchymal stem cells-derived ECM (BMSC-dECM) scaffold on the chondrogenic differentiation of marrow clots following microfracture in vitro. In this study, we manufactured the BMSC-dECM scaffold using a freeze-drying method. To obtain the marrow clots, a full-thickness cartilage defect was established and microholes were created in the trochlear groove of New Zealand white rabbits. The samples were divided and cultured in vitro for 1, 2, 4, and 8 weeks. The samples included a culture of the marrow clot alone (Group 1), a culture of the marrow clot with transforming growth factor-beta 3 (TGF-β3) (Group 2), a culture of the composite of the BMSC-dECM scaffold and the marrow clot alone (Group 3), and a culture of the composite with TGF-β3 (Group 4). A smooth and glossy surface was observed in Group 2 and Group 4 over time, but the surface for Group 4 was larger from week 1 onward. Compressive strength gradually increased in Groups 2 and 4, and greater increases were observed in Group 4 during the 8-week culture period. Enhanced cartilage-like matrix deposition of glycosaminoglycan (GAG) and type II collagen were confirmed by Safranin O and immunohistochemistry staining, respectively, in Groups 2 and 4. The GAG and collagen contents also gradually increased over time in Groups 2 and 4; the increase was greater in Group 4. In addition, real-time-polymerase chain reaction demonstrated that the expression of chondrogenic genes, such as COL2, ACAN, and SOX9, was gradually upregulated in Groups 2 and 4. However, greater increases in the expression of these cartilage-like genes were observed in Group 4 from week 4 onward. Our results suggest that the BMSC-dECM scaffold may favor the chondrogenesis of marrow clots following microfracture in vitro. In conclusion, these tissue engineering-like constructs could be potential candidates for cartilage repair.

Previous studies have shown that plastic compression (PC) of collagen gels allows a rapid and controlled fabrication of matrix- and cell-rich constructs in vitro that closely mimic the structure and characteristics of tissues in vivo. Microvascular endothelial cells, the major cell type making up the blood vessels in the body, were added to the PC collagen to determine whether cells attach, survive, grow, and express endothelial cell characteristics when seeded alone or in coculture with other cells. Endothelial cells seeded on the PC collagen containing human foreskin fibroblasts (HFF) or human osteoblasts (HOS) formed vessel-like structures over 3 weeks in culture without the addition of exogenous growth factors in the medium. In contrast, on the PC scaffolds without HFF or HOS, human dermal microvascular endothelial cells (HDMEC) exhibited a typical cobblestone morphology for 21 days under the same conditions. We propose that the coculture of primary endothelial cells with PC collagen constructs, containing a stromal cell population, is a valuable technique for in vitro modeling of proangiogenic responses toward such biomimetic constructs in vivo. A major observation in the cocultures was the absence of gel contraction, even after 3 weeks of fibroblast culture. This collagen form could, for example, be of great value in tissue engineering of the skin, as contractures are both aesthetically and functionally disabling.

Neural progenitor cells (NPCs) derived from pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced PSCs, are promising cell source for the treatment of various neurological diseases. NPC derivation from PSCs is regulated by microenvironment factors that influence cell fate via paracrine and autocrine effects. In this study, ESC-derived NPC aggregates were replated in the secretomes of bone marrow-derived human mesenchymal stem cells (hMSCs) generated under hypoxia or normoxia to investigate the effects of hMSC secretome on NPC cellular behaviors. The results demonstrated that hMSC secretomes stimulated endogenous secretion of extracellular matrices from NPC aggregates and enhanced cell adhesion and proliferation. NPC functional differentiation measured by migration length, neurite extension, and the yield of neural and glial cells were also increased by threefold to fourfold. Inhibition of fibroblast growth factor-2, transforming growth factor-β1, and brain-derived neurotrophic factor signaling differentially reduced the adherent cell number, migration length, and neurite extension, suggesting the regulatory effects of a broad spectrum of hMSC-derived factors. In summary, ESC-derived NPC aggregates in hypoxic hMSC secretomes may represent a suitable combination to promote the engraftment and neurogenesis in vivo.

We investigated the effects of CD14 macrophages and proinflammatory cytokines on chondrogenic differentiation of osteoarthritic synovium-derived stem cells (SDSCs). Osteoarthritic synovial fluid was analyzed for interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and IL-6. Levels of stem cell surface markers in osteoarthritic SDSCs were evaluated using flow cytometry. CD14-negative cells were obtained using magnetically activated cell sorting. We compared chondrogenic potentials between whole cells and CD14-negative cells in CD14^low cells and CD14^high cells, respectively. To assess whether nuclear factor-κB (NF-κB) and CCAAT/enhancer-binding protein β (C/EBPβ) modulate IL-1β-induced alterations in chondrogenic potential, we performed small interfering RNA transfection. We observed a significant correlation between the CD14 ratio in osteoarthritic SDSCs and IL-1β and TNF-α in osteoarthritic synovial fluid. Phenotypic characterization of whole cells and CD14-negative cells showed no significant differences in levels of stem cell markers. mRNA expression of type II collagen was higher in CD14-negative cell pellets than in whole cell pellets. Immunohistochemical staining indicated higher levels of type II collagen in the CD14-negative cell pellets of CD14^high cells than in whole cell pellets of CD14^high cells. As expected, IL-1β and TNF-α significantly inhibited the expression of chondrogenic-related genes in SDSCs, an effect which was antagonized by knockdown of NF-κB and C/EBPβ. Our results suggest that depletion of CD14⁺ synovial macrophages leads to improved chondrogenic potential in CD14^high cell populations in osteoarthritic SDSCs, and that NF-κB (RelA) and C/EBPβ are critical factors mediating IL-1β-induced suppression of the chondrogenic potential of human SDSCs.

Gene and cellular therapies are nowadays part of therapeutic strategies for the treatment of diverse pathologies. The drawbacks associated with gene therapy—low levels of transgene expression, vector loss during mitosis, and gene silencing—need to be addressed. The pEPI-1 and pEPito family of vectors was developed to overcome these limitations. It contains a scaffold/matrix attachment region, which anchors its replication to cell division in eukaryotic cells while in an extrachromosomal state and is less prone to silencing, due to a lower number of CpG motifs. Recent success showed that ocular gene therapy is an important tool for the treatment of several diseases, pending the overcome of the aforementioned limitations. To achieve sustained gene delivery in the retina, we evaluated several vectors based on pEPito and pEPI-1 for their ability to sustain transgene expression in retinal cells. These vectors stably transfected and replicated in retinal pigment epithelial (RPE) cells. Expression levels were promoter dependent with constitutive promoters cytomegalovirus immediate early promoter (CMV) and human CMV enhancer/human elongation factor 1 alpha promoter yielding the highest levels of transgene expression compared with the retina-specific RPE65 promoter. When injected in C57Bl6 mice, transgene expression was sustained for at least 32 days. Furthermore, the retina-specific RPE65 promoter showed higher efficiency in vivo compared to in vitro. In this study, we demonstrate that by combining tissue-specific promoters with a mitotic stable system, less susceptible to epigenetic silencing such as pEPito-based plasmids, we can achieve prolonged gene expression and a sustained therapeutic effect.

Tissue engineering endeavors to create replacement tissues and restore function that may be lost through infection, trauma, and cancer. However, wide clinical application of engineered scaffolds has yet to come to fruition due to inadequate vascularization. Here, we fabricate hydrogel constructs using Pluronic^® F127 as a sacrificial microfiber, creating microchannels within biocompatible, biodegradable type I collagen matrices. Microchannels were seeded with human umbilical vein endothelial cells (HUVEC) or HUVEC and human aortic smooth muscle cells (HASMC) in co-culture, generating constructs with an internal endothelialized microchannel. Histological analysis demonstrated HASMC/HUVEC-seeded constructs with a confluent lining after 7 days with preservation and further maturation of the lining after 14 days. Immunohistochemical staining demonstrated von Willebrand factor and CD31⁺ endothelial cells along the luminal surface (neointima) and alpha-smooth muscle actin expressing smooth muscle cells in the subendothelial plane (neomedia). Additionally, the deposition of extracellular matrix (ECM) components, heparan sulfate and basal lamina collagen IV were detected after 14 days of culture. HUVEC-only- and HASMC/HUVEC-seeded microchannel-containing constructs were microsurgically anastomosed to rat femoral artery and vein and perfused, in vivo. Both HUVEC only and HUVEC/HAMSC-seeded constructs withstood physiologic perfusion pressures while their channels maintained their internal infrastructure. In conclusion, we have synthesized and performed microvascular anastomosis of tissue-engineered hydrogel constructs. This represents a significant advancement toward the generation of vascularized tissues and brings us closer to the fabrication of more complex tissues and solid organs for clinical application.

We assessed the effects of oxygen tension on lubricin expression in bovine chondrocytes and cartilage explants and a role for hypoxia-inducible transcription factor (HIF)-1α in regulating lubricin expression was investigated using a murine chondroprogenitor cell line, ATDC5, and bovine chondrocytes isolated from superficial and middle/deep zones of femoral cartilage. ATDC5 cells and bovine chondrocytes were cultured in micromass under different oxygen tensions (21%, 5%, and 1%). ATDC5 cells and middle/deep zone chondrocytes that initially had low lubricin expression levels were also cultured with or without transforming growth factor (TGF)-β1. Quantitative reverse transcription (RT)-PCR was used to determine lubricin and chondrogenic marker gene mRNA levels and immunohistochemistry was used to assess lubricin protein expression. Explant cartilage plugs cultured under different oxygen tensions were also subjected to immunohistological analysis for lubricin. HIF-1α gene silencing was achieved by electroporatic transfer into ATDC5 cells. A low oxygen tension reduced lubricin gene expression levels in bovine superficial chondrocytes, TGF-β1-treated middle/deep zone chondrocytes, and TGF-β1-treated ATDC5 cells. Lubricin expression in explant cartilage was also suppressed under hypoxia. HIF-1α gene silencing in ATDC5 cells attenuated the lubricin expression response to the oxygen tension. These results corroborate with previous studies that the oxygen tension regulates lubricin gene expression and suggest that HIF-1α plays an important role in this regulation. The normal distribution of lubricin in articular cartilage may be due to the hypoxic oxygen environment of cartilage as it is an avascular tissue. An oxygen tension gradient may be a key factor for engineering cartilage tissue with a layered morphology.

Keloids are locally exuberant dermal scars characterized by excessive fibroblast proliferation and matrix accumulation. Although treatment strategies include surgical removal and intralesional steroid injections, an effective regimen is yet to be established due to a high rate of recurrence. The regressing center and growing margin of the keloid have different collagen architecture and also differ in the rate of proliferation. To investigate whether proliferation is responsive to collagen topography, keloid, scar, and dermal fibroblasts were cultured on nanopatterned scaffolds varying in collagen fibril diameter and alignment-small and large diameter, aligned and random fibrils, and compared to cells grown on flat collagen-coated substrates, respectively. Cell morphology, proliferation, and expression of six genes related to proliferation (cyclin D1), phenotype (α-smooth muscle actin), and matrix synthesis (collagens I and III, and matrix metalloproteinase-1 and -2) were measured to evaluate cell response. Fibril alignment was shown to reduce proliferation and matrix synthesis in all three types of fibroblasts. Further, keloid cells were found to be most responsive to nanotopography.

The secretion of trophic factors that promote angiogenesis from mesenchymal stem cells (MSCs) is a promising cell-based therapeutic treatment. However, clinical efficacy has proved variable, likely on account of ill-defined cell delivery formulations and the inherent complexity of cellular secretion. Here we show how controlling the mechanical properties and protein composition of the extracellular matrix (ECM) surrounding MSCs can guide proangiogenic signaling. Conditioned media from MSCs adherent to polyacrylamide hydrogel functionalized with fibronectin, collagen I, or laminin was applied to 3D matrigel cultures containing human microvascular endothelial cells (HMVECs). The degree of tubulogenesis in HMVECs is shown to depend on both the substrate rigidity and matrix protein composition. MSCs cultured on fibronectin-modified hydrogels show a stiffness dependence in proangiogenic signaling with maximum influence on tubulogenesis observed from 40 kPa conditioned media, twofold higher than commercially available cocktails of growth factors. Quantitative real-time–polymerase chain reaction reveals stiffness-dependent expression of multiple factors involved in angiogenesis that corroborate the functional tubulogenesis assay. Restricting cell spreading with micropatterned surfaces attenuates the conditioned media effects; however, small-molecule inhibitors of actomyosin contractility do not significantly reduce the functional outcome. This work demonstrates how controlling matrix rigidity and protein composition can influence the secretory profile of MSCs. Model systems that deconstruct the physical and biochemical cues involved in MSC secretion may assist in the design of hydrogel biomaterials for cell-based therapies.

Autologous engineered skin (ES) offers promise as a treatment for massive full thickness burns. Unfortunately, ES is orders of magnitude weaker than normal human skin causing it to be difficult to apply surgically and subject to damage by mechanical shear in the early phases of engraftment. In addition, no manufacturing strategy has been developed to tune ES biomechanics to approximate the native biomechanics at different anatomic locations. To enhance and tune ES biomechanics, a coaxial (CoA) electrospun scaffold platform was developed from polycaprolactone (PCL, core) and gelatin (shell). The ability of the coaxial fiber core diameter to control both scaffold and tissue mechanics was investigated along with the ability of the gelatin shell to facilitate cell adhesion and skin development compared to pure gelatin, pure PCL, and a gelatin-PCL blended fiber scaffold. CoA ES exhibited increased cellular adhesion and metabolism versus PCL alone or gelatin-PCL blend and promoted the development of well stratified skin with a dense dermal layer and a differentiated epidermal layer. Biomechanics of the scaffold and ES scaled linearly with core diameter suggesting that this scaffold platform could be utilized to tailor ES mechanics for their intended grafting site and reduce graft damage in vitro and in vivo.

Extracellular matrix (ECM) has been utilized as a biological scaffold for tissue engineering applications in a variety of body systems, due to its bioactivity and biocompatibility. In the current study we developed a modified protocol for the efficient and reproducible derivation of mesenchymal progenitor cells (MPCs) from human embryonic stem cells as well as human induced pluripotent stem cells (hiPSCs) originating from hair follicle keratinocytes (HFKTs). ECM was produced from these MPCs and characterized in comparison to adipose mesenchymal stem cell ECM, demonstrating robust ECM generation by the excised HFKT-iPSC-MPCs. Exploiting the advantages of electrospinning we generated two types of electrospun biodegradable nanofiber layers (NFLs), fabricated from polycaprolactone (PCL) and poly(lactic-co-glycolic acid) (PLGA), which provide mechanical support for cell seeding and ECM generation. Elucidating the optimized decellularization treatment we were able to generate an available “off-the-shelf” implantable product (NFL-ECM). Using rat subcutaneous transplantation model we demonstrate that this stem-cell-derived construct is biocompatible and biodegradable and holds great potential for tissue regeneration applications.

Mesenchymal stem cell (MSC) loaded bio-scaffold transplantation is a promising therapeutic approach for bone regeneration and repair. However, growing evidence shows that pro-inflammatory mediators from injured tissues suppress osteogenic differentiation and impair bone formation. To improve MSC-based bone regeneration, it is important to understand the mechanism of inflammation mediated osteogenic suppression. In the present study, we found that synovial fluid from rheumatoid arthritis patients and pro-inflammatory cytokines including interleukin-1α, interleukin-1β, and tumor necrosis factor α, stimulated intercellular adhesion molecule-1(ICAM-1) expression and impaired osteogenic differentiation of MSCs. Interestingly, overexpression of ICAM-1 in MSCs using a genetic approach also inhibited osteogenesis. In contrast, ICAM-1 knockdown significantly reversed the osteogenic suppression. In addition, after transplanting a traceable MSC-poly(lactic-co-glycolic acid) construct in rat calvarial defects, we found that ICAM-1 suppressed MSC osteogenic differentiation and matrix mineralization in vivo. Mechanistically, we found that ICAM-1 enhances MSC proliferation but causes stem cell marker loss. Furthermore, overexpression of ICAM-1 stably activated the MAPK and NF-κB pathways but suppressed the PI3K/AKT pathway in MSCs. More importantly, specific inhibition of the ERK/MAPK and NF-κB pathways or activation of the PI3K/AKT pathway partially rescued osteogenic differentiation, while inhibition of the p38/MAPK and PI3K/AKT pathway caused more serious osteogenic suppression. In summary, our findings reveal a novel function of ICAM-1 in osteogenesis and suggest a new molecular target to improve bone regeneration and repair in inflammatory microenvironments.

In this study, we have reported the incorporation of a multi-modal contrast agent based on hydroxyapatite nanocrystals, within a poly(caprolactone)(PCL) nanofibrous scaffold by electrospinning. The multifunctional hydroxyapatite nanoparticles (MF-nHAp) showed simultaneous contrast enhancement for three major molecular imaging techniques. In this article, the magnetic resonance (MR) contrast enhancement ability of the MF-nHAp was exploited for the purpose of potentially monitoring as well as for influencing tissue regeneration. These MF-nHAp containing PCL scaffolds were engineered in order to enhance the osteogenic potential as well as its MR functionality for their application in bone tissue engineering. The nano-composite scaffolds along with pristine PCL were evaluated physico-chemically and biologically in vitro, in the presence of human mesenchymal stem cells (hMSCs). The incorporation of 30–40 nm sized MF-nHAp within the nanofibers showed a substantial increase in scaffold strength, protein adsorption, proliferation, and osteogenic differentiation of hMSCs along with enhanced MR functionality. This preliminary study was performed to eventually exploit the MR contrast imaging capability of MF-nHAp in nanofibrous scaffolds for real-time imaging of the changes in the tissue engineered construct.

The use of short interfering RNA (siRNA) in combination with stem cells and biocompatible scaffolds is a promising strategy in regenerative medicine. Our experimental strategy was to explore the possibility of forcing or guiding the chondrogenic differentiation of human mesenchymal stem cells (hMSCs) by knocking down a negative regulator of chondrogenesis, Slug transcription factor (TF), thus altering cell behavior. We found that TGFβ-driven chondrogenic differentiation of hMSCs cultured onto a hyaluronan-based scaffold, HYAFF^®-11, was strengthened after cell exposure to siRNA against Slug. Slug silencing was effective in promoting the expression of chondrogenic markers, including Col2A1, aggrecan, Sox9, LEF1, and TRPS1. In addition, we confirmed that HYAFF-11 is a good scaffold candidate for hMSC use in tissue engineering applications, and showed that it is effective in sustaining TGFβ3 treatment associated with a specific gene silencing. Interestingly, preliminary results from the experimental model described here suggested that, even in the absence of differentiation supplements, Slug silencing showed a pro-chondrogenic effect, highlighting both its potential use as an alternative to TGFβ treatment, and the critical role of the Slug TF in determining the fate of hMSCs.