Tissue engineering: from the bedside to the bench and back to the bedside.

The field of Tissue Engineering and Regenerative Medicine has evolved rapidly over the past thirty years. This review will summarize its history, current status and direction through the lens of clinical need, its progress through science in the laboratory and application back into patients. We can take pride in the fact that much effort and progress began with the surgical problems of children and that many surgeons in the pediatric surgical specialties have become pioneers and investigators in this new field of science, engineering, and medicine. Although the field has yet to fulfill its great promise, there have been several examples where a therapy has progressed from the first idea to human application within a short span of time and, in many cases, it has been applied in the surgical care of children.

Rapid isolation of bone marrow mesenchymal stromal cells using integrated centrifuge-based technology.

BACKGROUND AIMS: The use of bone marrow-derived mesenchymal stromal cells (MSCs) in cell-based therapies is currently being developed for a number of diseases. Thus far, the clinical results have been inconclusive and variable, in part because of the variety of cell isolation procedures and culture conditions used in each study. A new isolation technique that streamlines the method of concentration and demands less time and attention could provide clinical and economic advantages compared with current methodologies. In this study, we evaluated the concentrating capability of an integrated centrifuge-based technology compared with standard Ficoll isolation. METHODS: MSCs were concentrated from bone marrow aspirate using the new device and the Ficoll method. The isolation capabilities of the device and the growth characteristics, secretome production, and differentiation capacity of the derived cells were determined. RESULTS: The new MSC isolation device concentrated the bone marrow in 90 seconds and resulted in a mononuclear cell yield 10-fold higher and with a twofold increase in cell retention compared with Ficoll. The cells isolated using the device were shown to exhibit similar morphology and functional activity as assessed by growth curves and secretome production compared to the Ficoll-isolated cells. The surface marker and trilineage differentiation profile of the device-isolated cells was consistent with the known profile of MSCs. DISCUSSION: The faster time to isolation and greater cell yield of the integrated centrifuge-based technology may make this an improved approach for MSC isolation from bone marrow aspirates.

Ovine model for auricular reconstruction: porous polyethylene implants.

OBJECTIVES: We developed a large animal model for auricular reconstruction with engineered cartilage frameworks and evaluated the performance of porous polyethylene auricular implants in this model. METHODS: Eighteen high-density porous polyethylene auricular frameworks were implanted subcutaneously in the infra-auricular areas of 9 sheep. The implants were harvested 17 weeks later for gross and histologic examination. The perioperative and postoperative courses were carefully documented. RESULTS: Five implants became exposed, and 2 implants needed to be removed at 7 weeks. Additionally, 1 infected implant was removed at 2 weeks. Seromas developed in 2 implants because of drain failures and were drained successfully during the first postoperative week. There were no other surgical site complications. The remaining 10 implants had an acceptable cosmetic appearance at 17 weeks. CONCLUSIONS: The perioperative complication rate in the ovine porous polyethylene auricular implant model was higher than that reported for auricular reconstructions in humans. The implant exposures were likely caused by ischemia and excessive stress on the thin overlying skin, because vascularized flap coverage was not used. The histologic findings were comparable to the results reported for other animal models. This large animal model is appropriate for auricular reconstruction experiments, including engineered constructs.

Engineered vascularized bone grafts.

Clinical protocols utilize bone marrow to seed synthetic and decellularized allogeneic bone grafts for enhancement of scaffold remodeling and fusion. Marrow-derived cytokines induce host neovascularization at the graft surface, but hypoxic conditions cause cell death at the core. Addition of cellular components that generate an extensive primitive plexus-like vascular network that would perfuse the entire scaffold upon anastomosis could potentially yield significantly higher-quality grafts. We used a mouse model to develop a two-stage protocol for generating vascularized bone grafts using mesenchymal stem cells (hMSCs) from human bone marrow and umbilical cord-derived endothelial cells. The endothelial cells formed tube-like structures and subsequently networks throughout the bone scaffold 4-7 days after implantation. hMSCs were essential for stable vasculature both in vitro and in vivo; however, contrary to expectations, vasculature derived from hMSCs briefly cultured in medium designed to maintain a proliferative, nondifferentiated state was more extensive and stable than that with hMSCs with a TGF-beta-induced smooth muscle cell phenotype. Anastomosis occurred by day 11, with most hMSCs associating closely with the network. Although initially immature and highly permeable, at 4 weeks the network was mature. Initiation of scaffold mineralization had also occurred by this period. Some human-derived vessels were still present at 5 months, but the majority of the graft vasculature had been functionally remodeled with host cells. In conclusion, clinically relevant progenitor sources for pericytes and endothelial cells can serve to generate highly functional microvascular networks for tissue engineered bone grafts.

Growth factor directed chondrogenic differentiation of porcine bone marrow-derived progenitor cells.

BACKGROUND: Despite advances in surgical technique, reconstruction of a mandibular condyle still causes significant donor-site morbidity. The purpose of this study was to compare the effect of 3 different growth factors and define optimal cell culture conditions for bone marrow-derived progenitor cells to differentiate into chondrocytes for mandibular condyle reconstruction. METHODS: Porcine bone marrow-derived progenitor cells (pBMPCs) were cultured as a pellet for 2, 3, and 4 weeks under the following conditions: group 1, TGF-ß3 + standard medium; group 2, TGF-ß3 + BMP-2 + standard medium; group 3, TGF-ß3 + IGF-1 + standard medium; and group 4, TGF-ß3 + BMP-2 + IGF-1 + standard medium. Chondrogenic differentiation was evaluated using 3 lineage differentiation markers. RESULTS: The mean type II collagen positive area increased over weeks 2, 3, and 4 in group 4 compared to all the other groups (ANOVA; P = 0.005). At week 4, there was significantly greater type II collagen production in group 4 compared to all the other groups (ANOVA; P = 0.003). The medium in group 4 produces the greatest amount of cartilage when compared to groups 1, 2, and 3, and that 4 weeks produces the greatest amount of type II collagen. CONCLUSIONS: The results of this study indicate that the most efficacious medium for chondrogenic differentiation of pBMPCs was group 4 medium and the most type II collagen was produced at 4 weeks.

Rodent Model for Orthotopic Implantation of Engineered Liver Devices.

This study presents a novel surgical model developed to provide hematological support for implanted cellularized devices augmenting or replacing liver tissue function. Advances in bioengineering provide tools and materials to create living tissue replacements designed to restore that lost to disease, trauma, or congenital deformity. Such substitutes are often assembled and matured in vitro and need an immediate blood supply upon implantation, necessitating the development of supporting protocols. Animal translational models are required for continued development of engineered structures before clinical implementation, with rodent models often playing an essential early role. Our long-term goal has been generation of living tissue to provide liver function, utilizing advances in additive manufacturing technology to create 3D structures with intrinsic micron to millimeter scale channels modeled on natural vasculature. The surgical protocol developed enables testing various design iterations in vivo by anastomosis to the host rat vasculature. Lobation of rodent liver facilitates partial hepatectomy and repurposing the remaining vasculature to support implanted engineered tissue. Removal of the left lateral lobe exposes the underlying hepatic vasculature and can create space for a device. A shunt is created from the left portal vein to the left hepatic vein by cannulating each with separate silicone tubing. The device is then integrated into the shunt by connecting its inflow and outflow ports to the tubing and reestablishing blood flow. Sustained anticoagulation is maintained with an implanted osmotic pump. In our studies, animals were freely mobile after implantation; devices remained patent while maintaining blood flow through their millifluidic channels. This vascular anastomosis model has been greatly refined during the process of performing over 200 implantation procedures. We anticipate that the model described herein will find utility in developing preclinical translational protocols for evaluation of engineered liver tissue. Impact statement Tissue and organ transplantation are often the best clinically effective treatments for a variety of human ailments. However, the availability of suitable donor organs remains a critical problem. Advances in biotechnology hold potential in alleviating shortages, yet further work is required to surgically integrate large engineered tissues to host vasculature. Improved animal models such as the one described are valuable tools to support continued development and evaluation of novel therapies.

Three-dimensionally printed polycaprolactone and ß-tricalcium phosphate scaffolds for bone tissue engineering: an in vitro study.

PURPOSE: The purpose of this study was to evaluate porcine bone marrow-derived progenitor cell (pBMPC) proliferation and penetration into a novel 3-dimensionally printed scaffold. MATERIALS AND METHODS: Four different tissue engineering scaffolds to evaluate pBMPC proliferation and penetration were examined. Scaffolds were fabricated from polycaprolactone (PCL) or the combination of ß-tricalcium phosphate (ß-TCP) and PCL (50:50), with 2 separate channel sizes (1 mm [small (S)] vs 2 mm [large (L)]). Scaffolds were fabricated into 20 × 20 × 7-mm blocks by use of a TheriForm machine (Integra Life Sciences, Akron, OH). Four groups of scaffolds were examined for pBMPC proliferation and penetration: group 1, ß-TCP/PCL S; group 2, ß-TCP/PCL L; group 3, PCL S; and group 4, PCL L. Nonparametric mean (Kruskal-Wallis) and multiple comparisons tests were used to compare the 4 groups. RESULTS: No shrinkage or deformation was noted in any of the scaffold groups after 2 weeks of culture. Mean surface cell counts ranged from 13.4 to 87.8 cells/0.57 mm(2), with group 1 (ß-TCP/PCL S) having statistically significantly higher counts than the other groups (P < .001). Mean interior cell counts ranged from 10.9 to 75.6 cells/0.57 mm(2), with group 1 having the greatest interior cell count (P < .001). Total collagen formation ranged from 0.2% to 86%, with group 1 having the highest collagen formation (P < .001). CONCLUSIONS: The 3-dimensionally printed scaffold (ß-TCP/PCL) with 1-mm channels showed greater cellular proliferation, penetration, and collagen formation after a 2-week in vitro culture than the other scaffolds evaluated. ß-TCP/PCL S scaffolds warrant further evaluation for bone tissue engineering in vivo.

Our Better Angels and the Invention of Hope.

This 2020 Presidential Address was given at the American Pediatric Surgical Association 2021 Virtual Annual Meeting, May 20-22, 2021.

Liver-assist device with a microfluidics-based vascular bed in an animal model.

OBJECTIVE: This study evaluates a novel liver-assist device platform with a microfluidics-modeled vascular network in a femoral arteriovenous shunt in rats. SUMMARY OF BACKGROUND DATA: Liver-assist devices in clinical trials that use pumps to force separated plasma through packed beds of parenchymal cells exhibited significant necrosis with a negative impact on function. METHODS: Microelectromechanical systems technology was used to design and fabricate a liver-assist device with a vascular network that supports a hepatic parenchymal compartment through a nanoporous membrane. Sixteen devices with rat primary hepatocytes and 12 with human HepG2/C3A cells were tested in athymic rats in a femoral arteriovenous shunt model. Several parenchymal tube configurations were evaluated for pressure profile and cell survival. The blood flow pattern and perfusion status of the devices was examined by laser Doppler scanning. Cell viability and serum protein secretion functions were assessed. RESULTS: Femoral arteriovenous shunt was successfully established in all animals. Blood flow was homogeneous through the vascular bed and replicated native flow patterns. Survival of seeded liver cells was highly dependent on parenchymal chamber pressures. The tube configuration that generated the lowest pressure supported excellent cell survival and function. CONCLUSIONS: This device is the first to incorporate a microfluidics network in the systemic circulatory system. The microvascular network supported viability and function of liver cells in a short-term ex vivo model. Parenchymal chamber pressure generated in an arteriovenous shunt model is a critical parameter that affects viability and must be considered in future designs. The microfluidics-based vascular network is a promising platform for generating a large-scale medical device capable of augmenting liver function in a clinical setting.

Tissue engineered hybrid tooth-bone constructs.

Proper rehabilitation of craniofacial defects is challenging because of the complexity of the anatomy and the component tissue types. The ability to simultaneously coordinate the regeneration of multiple tissues would make reconstruction more efficient and might reduce morbidity and improve outcomes. The craniofacial complex is unique because of the presence of teeth, in addition to skin, bone, cartilage, muscle, vascular, and neural tissues since teeth naturally grow in coordination with the craniofacial skeleton, our group developed an autologous, tooth-bone hybrid model to facilitate repair of mandibular defects in the Yucatan minipig. The hybrid tooth-bone construct was prepared by combining tooth bud cell-seeded scaffolds with autologous iliac crest bone marrow derived stem cell-seeded scaffolds, which were transplanted back into surgically created mandibular defects in the same minipig. The constructs were harvested after 12 and 20 weeks of growth. The resulting bone/tooth constructs were evaluated by X-ray, ultra high-resolution volume computed tomography (VCT), histological, immunohistochemical analyses, and transmission electron microscopy (TEM). The observed formation of small tooth-like structures consisting of organized dentin, enamel, pulp, cementum, periodontal ligament, and surrounded by regenerated alveolar bone, suggests the feasibility for regeneration of teeth and associated alveolar bone, in a single procedure. This model provides an accessible method for future clinical applications in humans.

Mechanical dissociation of swine liver to produce organoid units for tissue engineering and in vitro disease modeling.

The complex intricate architecture of the liver is crucial to hepatic function. Standard protocols used for enzymatic digestion to isolate hepatocytes destroy tissue structure and result in significant loss of synthetic, metabolic, and detoxification processes. We describe a process using mechanical dissociation to generate hepatic organoids with preserved intrinsic tissue architecture from swine liver. Oxygen-supplemented perfusion culture better preserved organoid viability, morphology, serum protein synthesis, and urea production, compared with standard and oxygen-supplemented static culture. Hepatic organoids offer an alternative source for hepatic assist devices, engineered liver, disease modeling, and xenobiotic testing.

Effect of ibuprofen on osteoblast differentiation of porcine bone marrow-derived progenitor cells.

PURPOSE: Nonsteroidal anti-inflammatory drugs are commonly prescribed to reduce inflammation and pain. However, little is known about the direct effect of these drugs on the differentiation of bone marrow-derived progenitor cells into osteoblasts. The purpose of this study was to determine the effect of ibuprofen on osteoblast differentiation and proliferation in a minipig model. MATERIALS AND METHODS: Bone marrow was aspirated from the minipig ilium, and porcine bone marrow-derived progenitor cells (pBMPCs) were isolated and expanded in standard culture medium. The pBMPCs were replated and differentiated into osteoblasts by use of osteogenic supplements (OS). Five groups were studied: negative control--pBMPCs in standard medium only; positive control--pBMPCs, standard culture medium, and OS; and 3 experimental groups--pBMPCs, standard culture medium, OS, and ibuprofen added in doses of 0.1, 1.0, and 3.0 mmol/L. Cell cultures were evaluated quantitatively by alkaline phosphatase (ALP) stain, von Kossa stain, and deoxyribonucleic acid (DNA) content. RESULTS: pBMPCs cultured with OS and low-dose ibuprofen (0.1 mmol/L) showed ALP stain, von Kossa stain, and DNA content similar to pBMPCs cultured in OS (positive control). pBMPCs cultured in higher doses of ibuprofen (1.0 and 3.0 mmol/L) produced significantly less positive staining of ALP and von Kossa and decreased DNA content. CONCLUSION: The results indicate that high-dose ibuprofen has a deleterious effect on pBMPC differentiation into osteoblasts whereas low-dose ibuprofen does not. The low dose of 0.1 mmol/L is the typical serum level when prescribed for clinical use.

Reconstructing mandibular defects using autologous tissue-engineered tooth and bone constructs.

PURPOSE: Current strategies for jaw reconstruction require multiple operations to replace bone and teeth. To improve on these methods, we investigated simultaneous mandibular and tooth reconstruction, using a Yucatan minipig model. MATERIALS AND METHODS: Tooth and bone constructs were prepared from third molar tooth tissue and iliac-crest bone marrow-derived osteoblasts isolated from, and implanted back into, the same pig as an autologous reconstruction. Implants were harvested after 12 and 20 weeks and evaluated by x-ray, ultrahigh-resolution volume computed tomographic (VCT), histological, and immunohistochemical analyses. RESULTS: Small tooth structures were identified, and consisted of organized dentin, enamel, pulp, and periodontal ligament tissues, surrounded by new bone. No dental tissues formed in implants without tooth-bud cells, and bone regeneration was observed to a limited extent. Immunohistochemical analyses using tooth-specific and bone-specific antibodies confirmed the identity of regenerated tissues. CONCLUSIONS: This pilot study supports the feasibility of tissue-engineering approaches for coordinated autologous tooth and mandible reconstruction, and provides a basis for future improvement of this technique for eventual clinical use in humans.

Pulmonary tissue engineering using dual-compartment polymer scaffolds with integrated vascular tree.

OBJECTIVES: The persistent shortage of donor organs for lung transplantation illustrates the need for new strategies in organ replacement therapy. Pulmonary tissue engineering aims at developing viable hybrid tissue for patients with chronic respiratory failure. METHODS: Dual-chamber polymer constructs that mimic the characteristics of the pulmonary air-blood interface were fabricated by microfabrication techniques using the biocompatible polymer polydimethylsiloxane. One compartment ("vascular chamber") was designed as a capillary network to mimic the pulmonary microvasculature. The other compartment ("parenchymal chamber") was designed to permit gas exchange. Immortalized mouse lung epithelium cells (MLE-12) were cultured on the surface of polystyrene microcarrier beads. These beads were subsequently injected into the parenchymal chamber of the dual-chamber microsystems. The vascular compartment was perfused with cell culture medium in a bioreactor and the construct was maintained in culture for 1 week. RESULTS: The microcarriers evenly distributed MLE-12 cells on the parenchymal compartment surface. Confluent cell layers were confirmed by fluorescent and electron microscopy. Adequate proliferation of MLE-12 cells within the construct was monitored via the DNA content. Viability of the cells was maintained over 1 week. Finally, cellular specificity and functional capacity in situ were demonstrated by immunostaining for proSP-B and proSP-C (alveolar epithelium), and by using MLE-12 cells transfected to overexpress green fluorescent protein. CONCLUSION: We conclude that functional hybrid microsystems mimicking the basic building plan of alveolar tissue can be engineered in vitro.

Correction to: Bone Marrow Derived Pluripotent Cells are Pericytes which Contribute to Vascularization.

Please note the following errors in the original version.

Decellularized extracellular matrix microparticles seeded with bone marrow mesenchymal stromal cells for the treatment of full-thickness cutaneous wounds.

Extracellular matrix materials mechanically dissociated into submillimeter particles have a larger surface area than sheet materials and enhanced cellular attachment. Decellularized porcine mesothelial extracellular matrix microparticles were seeded with bone marrow-derived mesenchymal stromal cells and cultured in a rotating bioreactor. The mesenchymal stromal cells attached and grew to confluency on the microparticles. The cell-seeded microparticles were then encapsulated in varying concentrations of fibrin glue, and the cells migrated rapidly off the microparticles. The combination of microparticles and mesenchymal stromal cells was then applied to a splinted full-thickness cutaneous in vivo wound model. There was evidence of increased cell infiltration and collagen deposition in mesenchymal stromal cells-treated wounds. Cell-seeded microparticles have potential as a cell delivery and paracrine therapy in impaired healing environments.

The promise of artificial liver replacement.

One of the most challenging clinical syndromes in medicine is that of acute liver failure (ALF). Many devices and systems have been devised to support ALF patients. This manuscript reviews the significant clinical findings of ALF, as well as, the non-biologic liver support systems and the bioartificial liver devices that have been clinically tested to support patients with this disease. Finally, we identify several improvements critical to the future of the field of bioartificial liver replacement therapy.

The effect of sustained delivery of vascular endothelial growth factor on angiogenesis in tissue-engineered intestine.

Our group has previously created a functional neointestine that is capable of restoring absorptive function. However, the endogenous level of vascular endothelial growth factor (VEGF) is markedly reduced in the construct compared to native bowel. Therefore, we wanted to locally deliver VEGF in a sustained fashion to upregulate angiogenesis in the neointestine. Rat recombinant VEGF was encapsulated in poly(lactide-co-glycolide) microspheres by a double emulsion method. Release kinetics and bioactivity were determined in vitro. Tissue-engineered intestine was generated by seeding donor neonatal rat intestinal organoid units onto a biodegradable polyglycolic acid scaffold along with VEGF-containing or empty microspheres, and wrapped in the omentum of recipient rats. After 4 weeks, the neointestinal cysts were analyzed for morphometry, VEGF levels, epithelial proliferation, and capillary density. Sustained release of biologically active VEGF was confirmed by in vitro studies. Intestinal constructs with VEGF microspheres were significantly larger than those containing empty microspheres. Tissue VEGF levels were significantly higher in neointestine loaded with encapsulated VEGF compared to those without growth factor. Epithelial cellular proliferation and capillary density were significantly increased in the VEGF-containing neointestinal constructs compared to empty constructs. Tissue-engineered intestine responds to sustained delivery of VEGF by upregulating microvasculature and epithelial proliferation.

Tissue-engineered spleen protects against overwhelming pneumococcal sepsis in a rodent model.

BACKGROUND/PURPOSE: Solid organs production is an ultimate goal of tissue engineering. After refining a technique for intestinal engineering, we applied it to a solid organ, the spleen. Overwhelming postsplenectomy sepsis results in death in nearly half of all cases. This risk is pronounced in children. Necrosis of autotransplanted spleen slices occurs prior to regeneration. We postulate that tissue engineering techniques might be superior. METHODS: Four groups of Lewis rats were compared: sham laparotomy, tissue-engineered spleen (TES), traditional spleen slices, and splenectomy. TES was generated from splenic units, multicellular components of juvenile spleen implanted on a biodegradable polymer scaffold, and spleen slices were derived from age-matched juveniles. Pneumococcal sepsis was induced at wk 16, and survival curves were constructed. RESULTS: Tissue-engineered spleen protected against pneumococcal septicemia with a survival proportion of 85.7% compared with 41.17% of splenectomized animals. Spleen slice was also protective with 71.43% survival. Compared with splenectomy, control and TES groups were statistically significant (P = 0.0002, P = 0.0087; hazard ratio of splenectomy = 5.493) and the Slice group was nearly statistically significant (P = 0.0642, hazard ratio of splenectomy = 2.673). CONCLUSIONS: TES is a novel application of tissue engineering to splenic regeneration and creates a functional spleen. This approach could be advantageous in severe pediatric trauma.