Tissue Engineering Part A: Research Advances

Intervertebral disc (IVD) degeneration has been implicated as a major component of spine pathology. As the IVD degenerates, the tissue becomes dehydrated, fibrotic, fissured, acellular, and calcified. These changes can lead to disc bulging, herniation, Schmorl's nodes, inflammation, and hyperinnervation. Injectable hydrogels have received much attention in recent years as scaffold for seeding cells to replenish disc cellularity and restore disc properties and function. However, they generally present poor mechanical properties. In this study, we investigate several novel thermosensitive chitosan hydrogels for their ability to mimic the mechanical properties of the nucleus pulposus (NP) tissue, while being injectable, able to entrap and maintain viability of NP cells, and retain matrix proteins. These new hydrogels were prepared by mixing chitosan (CH) with various combinations of three gelling agents: sodium hydrogen carbonate (SHC) and/or beta-glycerophosphate (BGP) and/or phosphate buffer (PB). The kinetics of gelation was studied at room and body temperature by rheology. Mechanical properties of the hydrogels were characterized under compression and torsion, and compared with human NP tissue. NP cells were seeded in the hydrogel when still liquid at room temperature, before its gelation at 37°C. Hydrogel cytocompatibility and functionality were assessed by measuring cell viability, metabolism, and proteoglycan synthesis. Although all the proposed hydrogels exhibited enhanced strength compared to CH-BGP thermosensitive hydrogels, and suitable cytocompatibility and rheological properties, one formulation (containing 2% chitosan, 7.5 mM of SHC, and 0.1 M of BGP) showed mechanical properties similar to human NP tissue, and stimulated better the synthesis and retention of proteoglycans from NP cells. Thus, this novel thermosensitive CH hydrogel shows promise for IVD regeneration.

This study developed a xeno-free (XF) spherical cell cluster of human adipose-derived stem cells (hASCs) for the treatment of severe ischemic diseases. hASCs are an attractive cell source for therapeutic angiogenesis. Herein, we describe an innovative method of culturing a three-dimensional cell mass (3DCM) in the XF condition and demonstrate the therapeutic potential and safety of XF-3DCMs in ischemic mice and in SCID/BALB/c Slc-nu/nu nude mice, respectively. XF-3DCMs were generated by culturing hASCs on a maltose-binding protein-linked basic fibroblast growth factor (bFGF)-immobilized polystyrene surface. XF-3DCMs produced comparable amounts of angiogenic factors such as bFGF, vascular endothelial growth factor, and interleukin-8 compared to 3DCMs formed in media containing FBS (fetal bovine serum)-3DCMs. The majority of the mice injected with XF-3DCMs exhibited limb salvaging and displayed similar blood perfusion compared to normal limbs. To a similar degree, as for FBS-3DCMs, XF-3DCMs enhanced therapeutic efficacy by increasing blood vessel regeneration, attenuating muscle degeneration and fibrosis, and facilitating the implantation of injected cells into host vessels. Furthermore, the soft agar colony-forming assay and in vivo tumorigenicity analysis showed that XF-3DCMs did not transform into tumor cells. No side effects were observed, including hematological and immunological toxicity, in mice injected with XF-3DCMs. These results suggest that XF-3DCMs show angiogenic and therapeutic efficacy in critical limb ischemia in mice and eliminate the safety problem of animal-derived material contamination, which is a prerequisite for clinical applications.

Platelet-rich plasma (PRP) and mesenchymal stem cells (MSCs) are promising tools for muscular-skeletal regeneration. However, increasing evidence has demonstrated controversial effects of PRP on the tissue regeneration. To obtain optimum PRP concentrations for MSC expansion and to accurately control osteogenic, adipocytic, and chondrogenic differentiation, MSCs were exposed to PRP alone or in combination with induction medium. We found that PRPs with the platelet concentration beyond 1500 × 10⁹ pl/L were preferable to promote MSC proliferation. In addition, PRPs ranging from 200 × 10⁹ to 3000 × 10⁹ pl/L were capable of augmenting MSC osteogenesis, and PRP with 1500 × 10⁹ pl/L was most effective for MSC osteogenic differentiation. Furthermore, PRPs in low platelet concentration range just slightly promoted MSC adipogenesis, and only when the platelet concentration was beyond 1800 × 10⁹ pl/L, the promoted effects were evident. Moreover, PRP range from 1000 to 3000 × 10⁹ pl/L significantly enhanced chondrogenesis of MSCs in the absence and presence of chondrogenic induction medium, and PRP with 2000 × 10⁹ pl/L was more effective for MSC chondrogenesis. Furthermore, we explored the mechanisms of PRP-induced MSC differentiation, showing that the growth factors played a major role in this process while other unknown factors may also be involved in it. At last, we measured the levels of cytokines to learn that PRP treatment suppressed the secretion of interleukin-1β (IL-1β), IL-6, and tumor necrosis factor-α, but favored the production of IL-10 by MSCs. In summary, our findings demonstrated that PRPs with different concentrations of platelets exerted different effects on proliferation and differentiation of MSCs, which indicated that preparing appropriate PRPs may be a precise and efficient strategy for improving MSC-based tissue regeneration.

The regeneration of cartilage is challenging due to its low metabolic rate and avascular nature. An effective treatment for osteochondral defects remains a clinical challenge. Glycosaminoglycans such as heparan sulfate (HS) bind and enhance the activity of prochondrogenic growth factors and thus hold potential for targeted tissue regeneration without the requirement for exogenous growth factors. In this study, we examine the use of a cell- and growth factor-free HS-based technique for osteochondral repair in a rabbit model. The binding affinity between HS and several reparative proteins (TGF-β1, BMP-2, FGF-2, PDGF-BB, and VEGF₁₆₅) was studied using surface plasmon resonance. Next, an HS-impregnated gel was implanted in a large osteochondral defect in the femoral trochlea of 19 New Zealand white rabbits to study the efficacy of the treatment. Over a 12-week period, HS showed significantly enhanced subchondral bone regeneration compared with a hydrogel control. Treatment with HS also resulted in an increased presence of hyaline cartilage in the chondral region. The use of HS in osteochondral defects appears to improve both subchondral and chondral tissue repair. Our data suggest that this effect is mediated by the ability of HS to promote endogenous growth factors.

Cell-based therapy is an ideal way of repairing defective cartilage. At present, the articular chondrocytes (ACs) is the cell source for cartilage repair. Problematically, in serial culture, the dedifferentiation of ACs occurs, which may result in graft failure. In the present study, we examined chondrogenic capacity and physical characteristics of proliferating layer chondrocytes (PLCs) from the growth plate cartilage and evaluated its regenerative activity for cartilage repair. We found that PLCs preserved more chondrogenic phenotypes, such as polygonal appearances, whereas ACs appeared fibroblast-like after seventh passage. Profoundly, the ACs expressed higher apoptosis-related proteins, such as cleaved caspase-9 and cleaved caspase-3, than PLCs. Also, the PLCs have higher proliferation rate than ACs, and the cell doubling time is 20.9 h for PLCs and 29.5 h for ACs. Using flow cytometry, we demonstrated that 26.6% of PLCs entered the S-phase after 16 h serum re-addition to starved cells compared with 13.3% of ACs. Otherwise, col2a1, aggrecan, sox5, sox6, and sox9 mRNAs were significantly increased in PLCs compared with ACs; in contrast, the col1a and col10a1 mRNA expression level in PLCs was lower than that in ACs. The glycosaminoglycan content in PLCs was higher than that in ACs by the direct 1,9-dimethylmethylene blue assay. Histological and immunohistochemical evaluations have demonstrated that significantly more chondrogenic extracellular matrix was detected in the PLCs group compared with the ACs group after implantation in nude mice. Taken together, our data indicate the PLCs preserved much more chondrogenic phenotypes than ACs in vitro and in vivo. These results might imply that PLCs act as the better cell source for reapairing and regenerating growth plate and articular cartilage.

Meniscal lesions represent one of the most common orthopedic injuries, and the standard of care, partial meniscectomy, often leads to long-term joint deterioration. The current meniscal replacement devices fail to recreate the native meniscus biomechanics that may explain the uncertainty of their clinical efficacy. A biomechanically functional, collagen-hyaluronan infused, three-dimensional printed polymeric scaffold was implanted into 18 sheep for up to 24 weeks to assess the scaffold's fixation, cellular response, tissue generation, integration to the host tissue, and effect on the surrounding articular cartilage. The scaffolds were assessed via gross inspection, histology, immunofluorescence, and biochemical analysis and the articular cartilage was assessed via gross inspection and histology. Scaffolds were ingrown with cells that generated a dense fibrocartilage-like tissue with significant collagen and glycosaminoglycan deposition. The stability of the surgical fixation was variable, however, with three partially displaced and five completely displaced implants at 12 weeks and three anatomic, four partially displaced, and two completely displaced implants at 24 weeks. Those implants remaining in the anatomic position displayed markedly improved outcomes. There was no significant degeneration observed in the surrounding articular cartilage for any condition. This study demonstrated the scaffold induces fibrochondrocytic tissue ingrowth, integrates robustly, and continues to mature as late as 24 weeks, and the articular cartilage is not adversely changed. The surgical model and fixation method must be improved and longer time points need to be investigated to further determine the scaffold's efficacy and chondroprotective abilities.

Mixed multicellular spheroids containing two or more cell types can be used as tissue models. We previously showed that mixed spheroids containing NIH3T3 mouse fibroblasts and MIN6 mouse pancreatic β cells showed core–shell type distribution, with NIH3T3 cells being located in the core region surrounded by MIN6 cells. In the present study, we investigated mechanisms underlying the determination and regulation of cellular distribution in these spheroids. Time-lapse microscopy showed that randomly mixed NIH3T3 and MIN6 cells separated from each other and showed core–shell type distribution. Treatment of these cells with cellular migration inhibitors suggested that the core–shell type distribution was regulated by focal adhesion kinase (FAK) signal in NIH3T3 cells. Microwells coated with collagen I, an FAK ligand, were prepared to regulate cellular distribution by inducing an exogenous FAK signal from outside the spheroids. NIH3T3/MIN6 spheroids formed in the collagen I-coated microwells showed random cellular distribution, suggesting that FAK signal-dependent migration of NIH3T3 cells toward the core region mediated the core–shell type distribution. These results indicate that cellular distribution in NIH3T3/MIN6 spheroids can be regulated by altering FAK-mediated directional migration of NIH3T3 cells using collagen I-coated microwells.

Peripheral arterial disease causes blood vessel dysfunction that requires surgical intervention. Current surgical interventions employ synthetic or allogeneic vascular grafts, which offer biocompatible material solutions that are not able to regenerate or grow with the patient. Decellularized scaffolds have gained significant momentum in the past few years, since they have the potential to regenerate in the patient. The aim of this study was to investigate the effects of modified decellularization protocol on the biocompatibility and immunogenicity of allogeneic rat abdominal aorta in an orthotopic rat model. Native syngeneic Wistar (W) and allogeneic Dark Agouti (DA) aortas, together with decellularized allogeneic DA aortas, were assessed histologically, immunohistochemically, and biomechanically. The immunogenicity of the untreated and decellularized syngeneic and allogeneic grafts was assessed in W rats, implanted orthotopically. Following implantation for 6 weeks, the grafts were explanted and assessed for the presence of T cells and macrophages by immunohistochemistry, and for their biomechanical integrity and histoarchitecture. No obvious histoarchitectural differences were observed between the native W and DA aortas, with both presenting similar three-layered structures. Histological analysis of decellularized DA aortas did not reveal any remaining cells. Explanted native DA allografts showed media necrosis, partial elastic fiber degradation, and adventitia thickening, as well as infiltration by lymphocytes (CD3⁺, CD4⁺) and macrophages (CD68+) in the adventitia. The explanted decellularized DA allografts indicated reduced immune injury compared to the explanted native DA allografts. The explanted native W syngeneic grafts showed a mild immune response, with an intact media and no lymphocyte infiltration. The explanted native DA allografts showed significantly lower collagen phase slope than the decellularized DA allografts before implantation, and significantly higher thickness than the explanted decellularized DA allografts. The results indicated that the modified decellularization protocol did not affect significantly the mechanical and histological properties of the native DA rat aorta. Overall, the immune response was improved by decellularization. Native DA allografts induced an adverse immune response in W rats, whereas syngeneic W grafts showed good tissue integration

Subarachnoid hemorrhage (SAH) is associated with vasospasm that is refractory to pharmacologic intervention. SAH vasospasm is associated with decreased phosphorylation of intracellular heat shock protein (HSP)20 and increased phosphorylation of HSP27. Phosphorylated HSP20 (p-HSP20) is associated with vascular smooth muscle relaxation and phosphorylated HSP27 with contraction and impaired relaxation. This study was undertaken to modulate these key signaling molecules using intracellular delivery technologies to elucidate the effect of modulation on vasomotor tone of intact vascular tissues. Rat aorta was used as a model system to elucidate the impact of HSP20/HSP27 modulation on physiologic vasomotor responses. HSP20 expression was silenced through siRNA transfection utilizing an endosomolytic diblock copolymer. The diblock copolymer self-assembles into small (∼50 nm) serum stable micellar nanoparticles that effectively package siRNA and facilitate tissue penetration, cell uptake, endosomal escape, intracellular bioavailability, and tissue-level silencing of target gene expression. To increase HSP27 activity, recombinant HSP27 was fused to a cell permeant peptide domain to facilitate protein uptake and activity. Furthermore, a cell permeant peptide mimetic of p-HSP20 was delivered through formulation into nano-polyplexes using a pH-responsive endosomolytic polymer. The impact of these biomolecular modulations on physiologic vasomotor responses in intact tissue was then quantified in a muscle bath. Treatment of rat aorta with HSP20 siRNA polymeric nanocarriers decreased expression of HSP20 which was associated with enhanced contractile responses to phenylephrine (PE) and impaired relaxation responses to sodium nitroprusside. Delivery of recombinant HSP27 to the tissue was associated with increased contractile responses to PE. Treatment with a peptide mimetic of p-HSP20 resulted in decreased contractile responses to PE. These results demonstrate that manipulation of protein levels of the small heat shock phosphoproteins, HSP20 and HSP27, in intact vascular tissues is associated with changes in vasomotor responses. In particular, decreasing HSP20 expression and addition of exogenous HSP27 led to enhanced contractility and impaired relaxation. This biochemical signature is similar to the phenotype of SAH associated vasospasm and suggests that alterations in downstream signaling events may be responsible for SAH induced vasospasm being refractory to upstream, receptor-mediated pharmacologic interventions.

Stem cell fate specification is known to be regulated by mechanical cues such as cell–cell interaction and the rigidity of the extracellular matrix (ECM). At present, spheroid cultures of stem cells are conventional starting materials to generate organoids with tissue-like structures. However, the influence of ECM rigidity on stem cell mechanobiology has only been briefly explored in in vitro three-dimensional (3D) cultures, which presents better physiological relevance than monolayer cultures. To investigate how ECM rigidity acts on neural stem cells (NSCs) to drive specific biological processes, we performed live 3D-imaging on neurospheres in medium, in 1% and 1.5% agarose gel-medium matrix. In this study, we also report a novel method of 3D spheroid expansion stress quantification. Our results revealed that neurospheres in stiffer ECM exhibited higher stem cell ratio than those in medium. Moreover, we observed a negative correlation between the signal intensity of the NSC marker, SSEA-1 (CD15), and mean stresses exerted by the cell. This observation is implicative of a sparse actomyosin network in NSCs than nonstem cells. Based on these results, we propose that enrichment of NSCs or directed differentiation may be carried out by tailoring ECM rigidity and possibly its composition.

New cell and tissue sources are needed for the regenerative treatment of articular cartilage damage. Human induced pluripotent stem cells (hiPSCs) are an abundant cell source due to their self-renewal capacity. Hyaline cartilage tissue particles derived from hiPSCs (hiPS-Carts), 1–3 mm in diameter, are one candidate source that can be used for transplantation. When transplanted to fill the defects of articular cartilage, hiPS-Carts form a repair tissue by integrating with each other and with adjacent host tissue. In this study, we analyzed the integration capacity using an in vitro model and found that hiPS-Carts spontaneously integrate with each other in vitro. hiPS-Carts consist of cartilage at the center and perichondrium-like membrane that wraps around the cartilage. The integration started at the perichondrium-like membrane at around 1 week. Then, the integration progressed to the cartilage within 4–8 weeks. RNA sequencing analysis identified a higher expression of FGF18 in the perichondrium-like membrane in hiPS-Carts compared with the central cartilage. The addition of FGF18 to the model accelerated the integration of hiPS-Carts, whereas the addition of a FGFR inhibitor inhibited it. These results suggest that FGF18 secreted from the perichondrium-like membrane plays a role in the integration of hiPS-Carts. Understanding the integration mechanism of hiPS-Carts is expected to contribute to the realization of regenerative treatment for patients with articular cartilage damage.

In an effort to construct in vitro liver tissue models for drug screening, human induced pluripotent stem cell (hiPSC)-derived hepatocyte-like cells (iHeps) are a promising cell source alternative to primary human hepatocytes (PHHs). However, thus far, the functions of iHeps are significantly reduced when compared to those of PHHs. In this study, we aimed to determine the role of oxygen supply to the cell layer, which has not been investigated as thoroughly as other conditions, such as soluble factors, the extracellular matrix, or co-culture with nonparenchymal cells, in improving the hepatic function of iHeps. Polydimethylsiloxane (PDMS), which has a high oxygen permeability, was used for cell culture [O₂ (+)], in addition to conventional tissue culture-treated polystyrene [TCPS; O₂ (−)]. During endoderm differentiation, O₂ (+) culture resulted in reduced expression of HIF-1α. As a result, we observed higher expression of the endoderm marker (GATA4). After hepatic differentiation, iHeps generated in O₂ (+) culture showed about a threefold increase per cell in albumin production. Moreover, among the O₂ (+) culture conditions, cells differentiating at a physiological oxygen concentration showed increased expression of several mature hepatic marker genes (ALB, CYP3A4, and CYP1A2) and increased ratio of ALB-positive cells. In O₂ (+) culture, iHeps exhibited a per cell oxygen consumption rate two times higher than that observed in O₂ (−) culture. Furthermore, in sufficiently oxygen-supplied culture systems, cells showed a self-organized, multilayered configuration, and the DNA content in the culture was two to three times higher than that in O₂ (−) culture. Considering the amount of DNA and the ration of iHeps in each culture condition, we could achieve about 7.5 times higher per unit surface area productivity of iHeps in O₂ (+) culture. Therefore, these results demonstrate that direct oxygenation through a PDMS membrane enhances the maturation and efficient production of iHeps. Thus, improving oxygen supply to the cell layer is important for iHeps culture, and direct oxygenation through a PDMS membrane is a promising alternative to conventional TCPS plates for the maturation of iHeps in vitro.

The regeneration of functional osteochondral tissue is mired by the difficulty of recapitulating the zone-dependent differences in tissue composition and constituent cell phenotype. The objective of the current work is to study the ability of multidirectional zonal scaffolds to promote the differentiation of murine bone marrow stromal cells (BMSCs) and chondrocytes along the osteogenic and chondrogenic lineages, respectively, by analyzing dual or triple transgenic fluorescent reporter expression in vitro and in vivo. BMSCs containing fluorescent reporters for bone sialoprotein (BSP) and dentin matrix protein (DMP-1) were seeded on zonal scaffolds in vitro, where successful differentiation was determined from a continued progression of BSP to DMP1-exclusive fluorescence and was corroborated by gene expression and histological analyses. Zonal chondrogenesis was then separately studied using articular chondrocytes containing fluorescent reporters for types 1, 2, and 10 collagens. The majority of fluorescent cells (65.1%) displayed exclusive fluorescence of the type 2 collagen reporter, where a significant portion (27.7%) cofluoresced type 2 and type 10 collagen reporters, indicating a progression to hypertrophy in vitro. Zonal gene expression, histological, and immunohistochemical analyses revealed prehypertrophic chondrocytes embedded in zonally-organized matrix. Upon ectopic implantation, the prehypertrophic chondrocytes formed mineralized tissue through an endochondral ossification pathway. Fluorescent multireporter cells accurately monitored the differentiation behavior of BMSCs and articular chondrocytes on zonal scaffolds and revealed subtle differences in tissue and chondrocyte phenotype.

Extracellular matrix (ECsM) bioscaffolds have been successfully used to treat five esophageal adenocarcinoma (EAC) patients following resection of neoplastic mucosal tissue. The present study evaluated the in vitro effect of ECM harvested from nonmalignant, decellularized tissue on EAC cell phenotype to understand the molecular mechanisms underlying the clinical findings. Nonmalignant (Het-1A), metaplastic (CP-A), and neoplastic (SK-GT-4, OE33) esophageal epithelial cells were exposed to ECM degradation products (250 μg/mL) prepared from heterologous urinary bladder tissue or homologous esophageal mucosa tissue, and evaluated for cell morphology, cell function, and EAC signaling pathways. Both the ECM sources downregulated neoplastic cell phenotype, but had distinctive tissue-specific effects. Urinary bladder ECM decreased OE33 and SK-GT-4 metabolism and increased CP-A apoptosis. Esophageal ECM decreased SK-GT-4, CP-A, and Het-1A proliferation; robustly downregulated PI3K-Akt-mTOR, cell cycle/DNA replication signaling, and upregulated autophagy signaling in OE33 cells; and increased cell cycle/DNA replication signaling in Het-1A cells. Both ECM sources decreased OE33 proliferation and phosphorylated AKT in OE33 cells, and in contrast, increased phosphorylated AKT in Het-1A cells. The results support the concept that the biochemical signals in nonmalignant ECM can downregulate neoplastic cell phenotype with minimal, and sometimes opposite, effects on normal cells. PI3K-Akt signaling has been implicated in EAC progression and these ECM-mediated effects may be favorable for an esophageal therapy following cancer resection.

Construction of small and continuous capillary networks is a fundamental challenge for the development of three-dimensional (3D) tissue engineering. In particular, to construct mature and stable capillary networks, it is important to consider interactions between endothelial cells and pericytes. This study aimed to construct stable capillary networks covered by pericyte-like perivascular cells, which maintain the lumen of small diameter similar to that of capillary structures in vivo. Vascular sprouting, capillary extension, and stabilization were investigated using a 3D angiogenesis model containing human umbilical vein endothelial cells (HUVECs) and mesenchymal stem cells (MSCs) in a microfluidic device. A series of HUVEC:MSC ratios was tested; the ratio was found to be an important factor in the construction of capillary structures. We found that stable capillary networks that were covered by MSC-derived perivascular cells can be constructed at 1:1 HUVEC:MSC ratio. The constructed capillary networks had continuous lumens with <10-μm diameter, which were maintained for at least 21 days. This angiogenic process and basement membrane formation were regulated by HUVEC–MSC interactions.