Tissue Engineering Part A: Research Advances

Secondary alveolar bone grafts (ABGs) are the standard treatment for the alveolar defect in patients with cleft lip and palate (CLP), but remain invasive and have several disadvantages such as delayed timing of alveolar repair, donor-site complications, graft resorption, and need for multiple surgeries. Earlier management of the alveolar defect (primary ABG) would be ideal, but is limited by the minimal bony donor sites available in the infant. In this study we used a critical-size alveolar bone defect model in the rat to investigate the use of Wharton's Jelly (WJ), the stem cell-rich connective tissue matrix of the umbilical cord, to generate bone within the alveolar cleft. Human WJ was isolated and implanted into a critical-size alveolar bone defect model representative of secondary cleft ABG surgery in 10–11-week-old male Sprague–Dawley rats. The defects were monitored with CT imaging of living animals to evaluate bone regrowth and healing over 24 weeks, followed by histomorphometric evaluation at 24 weeks, after the last CT scan. CT data confirmed that the defect size was critical and did not lead to the union of the bones in the control animals (n = 12) for the entire duration of the study. New bone growth was stimulated leading to partial-to-full closure of the defect in the animals treated with WJ (n = 12). Twenty four weeks postoperatively, the percent increase in new bone formation in the WJ-treated group (156.58% ± 20.67%) was markedly higher than that in the control group (50.36% ± 21.07%) (p < 0.05). Histomorphometric data also revealed significantly greater new bone formation in WJ-treated versus control animals, confirming CT findings. qPCR analysis of human Alu elements was unable to detect any appreciable long-term persistence of human cells in the new bone, indicating that WJ may enhance bone growth by mediating osteoinduction in the host tissue, rather than through osteogenic differentiation of WJ-embedded cells.

Temporal and spatial presentations of biological cues are critical for tissue engineering. There is a great need in improving the incorporation of bioagent(s) (specifically growth factor(s) [GF(s)]) onto three-dimensional scaffolds. In this study, we developed a process to combine additive manufacturing (AM) technology with acoustic droplet ejection (ADE) technology to control GF distribution. More specifically, we implemented ADE to control the distribution of recombinant human bone morphogenetic protein-2 (rhBMP-2) onto polycaprolactone (PCL)-based tissue engineering constructs (TECs). Three substrates were used in this study: (1) succinimide-terminated PCL (PCL-N-hydroxysuccinimide [NHS]) as model material, (2) alkali-treated PCL (PCL-NaOH) as first control material, and (3) fibrin-coated PCL (PCL-Fibrin) as second control material. It was shown that our process enables a pattern of BMP-2 spots of ∼250 μm in diameter with ∼700 μm center-to-center spacing. An initial concentration of BMP-2 higher than 300 μg/L was required to retain a detectable amount of GF on the substrate after a wash with phosphate-buffered solution. However, to obtain detectable osteogenic differentiation of C2C12 cells, the initial concentration of BMP-2 higher than 750 μg/L was needed. The cells on PCL-NHS samples showed spatial alkaline phosphatase staining correlating with local patterns of BMP-2, although the intensity was lower than the controls (PCL-NaOH and PCL-Fibrin). Our results have demonstrated that the developed AM-ADE process holds great promise in creating TECs with highly controlled GF patterning.

In liver tissue engineering, cell culture in spheroids is now well recognized to promote the maintenance of hepatic functions. However, the process leading to spheroids formation is time consuming, costly, and not easy to scale-up for further use in human bioartificial liver (BAL) applications. In this study, we encapsulated HepaRG cells (precursors of hepatocyte-like cells) in 1.5% alginate beads without preforming spheroids. Starting from a given hepatic biomass, we analyzed cell differentiation and metabolic performance for further use in a fluidized-bed BAL. We observed that cells self-rearranged as aggregates within the beads and adequately differentiated over time, in the absence of any differentiating factors classically used. On day 14 postencapsulation, cells displayed a wide range of hepatic features necessary for the treatment of a patient in acute liver failure. These activities include albumin synthesis, ammonia and lactate detoxification, and the efficacy of the enzymes involved in the xenobiotic metabolism (such as CYP1A1/2).

In this study, we evaluated the ability of stretch-grown tissue-engineered nerve grafts (TENGs) to perform as a living scaffold for axonal regeneration across a severed spinal cord lesion. TENGs, consisting of stretch-grown axons spanning two populations of dorsal root ganglia neurons, have proven to be effective in bridging gaps in peripheral nerve injury. A complete transection was performed at the thoracic level in a rodent model and 5 mm of cord was completely removed. TENGs encapsulated in a collagen hydrogel were placed within the cavity and compared against a collagen only transplant. Through hematoxylin and eosin (H&E) staining and immunohistochemistry, we found that TENGs survived up to 6 weeks post-transplant, extending neuronal processes into and through host tissue early on in both the rostral and caudal direction. In several cases, TENG axons penetrated into and through glial scar tissue, appearing to overcome a common obstacle for axonal regeneration in spinal cord injuries (SCIs). H&E staining also provided evidence that animals treated with TENGs resulted in lesion sites with greater tissue infiltration and less compression than animals treated with a collagen hydrogel only, an encouraging finding given the severity of the injury model. We also observed effects the TENGs had on glial scar formation, cyst formation, and immune response at multiple time points as these are common difficulties faced in tissue engineering methods to treat or repair SCI. If able to address these universal challenges associated with SCI, TENGs may offer an alternative option in neural transplantation and may represent a viable tool in the multifaceted treatment of SCI.

Volumetric muscle loss (VML) resulting from extremity trauma presents functional deficits and fibrosis, ultimately manifesting disability. The extensive fibrotic accumulation is expected to interfere with neural, trophic, vascular, and mechanical connectivity of any possible regenerative medicine approaches. Our objective was to quantify the muscle properties and stiffness following injury and investigate if the fibrotic deposition could be mitigated using an antifibrotic agent; we hypothesized that antifibrotic treatment would prevent the overwhelming fibrotic response. Yorkshire Cross pigs (n = 10) were randomized to sham or a nontreated ∼20% VML injury. Immediately following surgery, injured animals were further randomized to nintedanib (Ofev; 300 mg/day) or no treatment for 30 days. Longitudinal analysis of muscle function via peroneal nerve stimulation, compartment volume, and quantitative muscle stiffness using shearwave elastography were conducted. Terminally comprehensive histopathologic, biochemical, and genetic investigations were conducted on the skeletal muscle and fibrosis. Through 4 weeks post-VML, nontreated muscles presented a significant deficit (23%) in maximal torque compared to the sham operated (p < 0.01). The stiffness in the VML defect area increased significantly (7-fold) in the VML-nontreated leg than the VML antifibrotic-treated legs by 4 weeks postinjury, which was coupled with the nontreated muscle having ∼40% more hydroxyproline per mg of tissue than those receiving antifibrotic treatment (p = 0.01). This work indicates that VML injury progressively induces fibrosis and muscle stiffness. Antifibrotic treatment can mitigate the pathologic development of fibrosis. Future work should evaluate optimal timing and duration of treatments combined with regenerative medicine approaches in efforts to improve function.

Osteoclasts are pivotal cells for bone remodeling and their activity is coordinated by osteocytes that reside inside the bone matrix. In vitro co-cultures of osteocytes and osteoclasts are therefore advantageous to analyze the crosstalk between these cell species. In this study, primary osteocytes were isolated from human bone in a multistep isolation process and embedded into three-dimensional collagen gels. Mature human osteoclasts were generated by differentiation of human peripheral blood mononuclear cells (PBMCs). Different surfaces were tested for osteoclast formation: suspension dishes, collagen gels, and normal tissue culture polystyrene. After detachment from the surfaces, osteoclasts showed typical morphology and gene expression of osteoclast markers. Osteoclasts that were differentiated on collagen exhibited the highest osteoclast marker expression. Cocultivation of mature osteoclasts with osteocytes was performed in a transwell system, with osteocytes, embedded in collagen gels at the apical side and osteoclasts on the basal side of a porous polyethylen terephtalate membrane, which allowed the separate gene expression analysis for osteocytes and osteoclasts. After 7 days of co-culture both cell species showed their typical morphology, which is multinucleated giant cells for osteoclasts and star-shaped cells with dendritic extensions for osteocytes. Furthermore, osteoclast markers tartrate-resistant acid phosphatase, carbonic anhydrase II, and cathepsin K were detected both on gene expression and protein level in single and co-cultures. Osteocytes showed gene expression of typical osteocyte markers E11, sclerostin, dentin matrix protein 1, osteocalcin, and receptor activator of nuclear factor-κ ligand both in single and co-culture.

Organoid and organ-on-a-chip have evolved as two critical but distinct approaches to develop human physiologically and pathologically relevant in vitro models. Although rapid progress has been witnessed in the past decade, there is no systematic comparison of their status and trends based on the scientometric analysis. In this study, we performed a comparative study of organoid and organ-on-a-chip using bibliometric methods. A total of 2790 documents published between 2009 and 2018 were retrieved and analyzed. Our results showed that both organoid and organ-on-a-chip had experienced rapid growth in their academic and social impacts and influenced a wide spectrum of disciplines, but with a major distinct focus on Cell Biology and Nanoscience Nanotechnology, respectively. The hotspots of organoid research were expanding from in vitro differentiation of Lgr5 stem cells to mechanistic studies of diseases, while the hotspots of the organ-on-a-chip research were transiting from the establishment of microfluidic devices for in vitro cell culture to stem cell differentiation and tissue engineering. Interestingly, there was a growing trend of combining organoid with organ-on-a-chip in the last few years. This comparative study presented a unique perspective to understand the evolutive history and future trends of organoid and organ-on-a-chip for emerging human relevant in vitro organotypic models.

One million estimated cases of spinal cord injury (SCI) have been reported in the United States and repairing an injury has constituted a difficult clinical challenge. The complex, dynamic, inhibitory microenvironment postinjury, which is characterized by proinflammatory signaling from invading leukocytes and lack of sufficient factors that promote axonal survival and elongation, limits regeneration. Herein, we investigated the delivery of polycistronic vectors, which have the potential to coexpress factors that target distinct barriers to regeneration, from a multiple channel poly(lactide-co-glycolide) (PLG) bridge to enhance spinal cord regeneration. In this study, we investigated polycistronic delivery of IL-10 that targets proinflammatory signaling, and NT-3 that targets axonal survival and elongation. A significant increase was observed in the density of regenerative macrophages for IL-10+NT-3 condition relative to conditions without IL-10. Furthermore, combined delivery of IL-10+NT-3 produced a significant increase of axonal density and notably myelinated axons compared with all other conditions. A significant increase in functional recovery was observed for IL-10+NT-3 delivery at 12 weeks postinjury that was positively correlated to oligodendrocyte myelinated axon density, suggesting oligodendrocyte-mediated myelination as an important target to improve functional recovery. These results further support the use of multiple channel PLG bridges as a growth supportive substrate and platform to deliver bioactive agents to modulate the SCI microenvironment and promote regeneration and functional recovery.