Maximizing neotissue growth kinetics in a perfusion bioreactor: An in silico strategy using model reduction and Bayesian optimization.

In regenerative medicine, computer models describing bioreactor processes can assist in designing optimal process conditions leading to robust and economically viable products. In this study, we started from a (3D) mechanistic model describing the growth of neotissue, comprised of cells, and extracellular matrix, in a perfusion bioreactor set-up influenced by the scaffold geometry, flow-induced shear stress, and a number of metabolic factors. Subsequently, we applied model reduction by reformulating the problem from a set of partial differential equations into a set of ordinary differential equations. Comparing the reduced model results to the mechanistic model results and to dedicated experimental results assesses the reduction step quality. The obtained homogenized model is 105 fold faster than the 3D version, allowing the application of rigorous optimization techniques. Bayesian optimization was applied to find the medium refreshment regime in terms of frequency and percentage of medium replaced that would maximize neotissue growth kinetics during 21 days of culture. The simulation results indicated that maximum neotissue growth will occur for a high frequency and medium replacement percentage, a finding that is corroborated by reports in the literature. This study demonstrates an in silico strategy for bioprocess optimization paying particular attention to the reduction of the associated computational cost.

Chitosan-Polyethylene Glycol Inspired Polyelectrolyte Complex Hydrogel Templates Favoring NEO-Tissue Formation for Cardiac Tissue Engineering.

Neo-tissue formation and host tissue regeneration determine the success of cardiac tissue engineering where functional hydrogel scaffolds act as cardiac (extracellular matrix) ECM mimic. Translationally, the hydrogel templates promoting neo-cardiac tissue formation are currently limited; however, they are highly demanding in cardiac tissue engineering. The current study focused on the development of a panel of four chitosan-based polyelectrolyte hydrogels as cardiac scaffolds facilitating neo-cardiac tissue formation to promote cardiac regeneration. Chitosan-PEG (CP), gelatin-chitosan-PEG (GCP), hyaluronic acid-chitosan-PEG (HACP), and combined CP (CoCP) polyelectrolyte hydrogels were engineered by solvent casting and assessed for physiochemical, thermal, electrical, biodegradable, mechanical, and biological properties. The CP, GCP, HACP, and CoCP hydrogels exhibited excellent porosity (4.24 ± 0.18, 13.089 ± 1.13, 12.53 ± 1.30 and 15.88 ± 1.10 for CP, GCP, HACP and CoCP, respectively), water profile, mechanical strength, and amphiphilicity suitable for cardiac tissue engineering. The hydrogels were hemocompatible as evident from the negligible hemolysis and RBC aggregation and increased adsorption of plasma albumin. The hydrogels were cytocompatible as evident from the increased viability by MTT (>94% for all the four hydrogels) assay and direct contact assay. Also, the hydrogels supported the adhesion, growth, spreading, and proliferation of H9c2 cells as unveiled by rhodamine staining. The hydrogels promoted neo-tissue formation that was proven using rat and swine myocardial tissue explant culture. Compared to GCP and CoCP, CP and HACP were superior owing to the cell viability, hemocompatibility, and conductance, resulting in the highest degree of cytoskeletal organization and neo-tissue formation. The physiochemical and biological performance of these hydrogels supported neo-cardiac tissue formation. Overall, the CP, GCP, HACP, and CoCP hydrogel systems promise novel translational opportunities in regenerative cardiology.

Angiogenic stimulation strategies in bone tissue regeneration.

Current tissue engineering strategies in bone repair and regeneration have limitations regarding tissue rejection, insufficient blood supply, and tissue integration. Specific host response results in isolation, degeneration, and subsequent loss of function of the implanted/scaffold biomaterial. Therefore, strategies to increase the interplay between angiogenesis and complex bone tissue formation are required to develop fully functional vascularized bone tissue. Angiogenesis is essential for oxygen/nutrient supply, waste removal, endothelial/stem cell homing, and the release of mitogenic/angiogenic/osteogenic factors. Hence, the challenge lies in understanding the complex interdependence of angiogenesis with neo-bone formation. Therefore, recent bone tissue regeneration strategies have focused on biomaterial development concerning induction of neovascularization and subsequent angiogenesis. Scaffold architecture (macro/micro/nano) scales, culture conditions (3-Dimension, hypoxia, etc), stimuli-dependent delivery of angiofactors, and gene delivery may significantly modulate vascularization in tissue-engineered products. Therefore, the current review discusses the key mechanisms/steps involved in defining the relationship between angiogenic and osteogenic factors. The recent strategies incorporating the above understanding in the development of bone tissue-engineered constructs are also deliberated. Eventually, these strategies may give the potential way forward to develop a bioengineered, vascularized bone tissue construct for implant applications.

Scaffold printing using biodegradable poly(1,4-butylene carbonate) ink: printability, in vivo physicochemical properties, and biocompatibility.

This study is the first to assess the applicability of biodegradable poly(1,4-butylene carbonate) (PBC) as a printing ink for fused deposition modeling (FDM). Here, PBC was successfully prepared via the bulk polycondensation of 1,4-butanediol and dimethyl carbonate. PBC was melted above 150°C in the heating chamber of an FDM printer, after which it flowed from the printing nozzle upon applying pressure and solidified at room temperature to create a three-dimensional (3D) scaffold structure. A 3D scaffold exactly matching the program design was obtained by controlling the temperature and pressure of the FDM printer. The compressive moduli of the printed PBC scaffold decreased as a function of implantation time. The printed PBC scaffold exhibited good in vitro biocompatibility, as well as in vivo neotissue formation and little host tissue response, which was proportional to the gradual biodegradation. Collectively, our findings demonstrated the feasibility of PBC as a suitable printing ink candidate for the creation of scaffolds via FDM printing.

Imatinib attenuates neotissue formation during vascular remodeling in an arterial bioresorbable vascular graft.

BACKGROUND: Bioresorbable vascular grafts (BVGs) can transform biologically into active blood vessels and represent an alternative to traditional synthetic conduits, which are prone to complications such as infection and thrombosis. Although platelet-derived growth factors and c-Kit positive cells play an important role in smooth muscle cell (SMC) migration and proliferation in vascular injury, atherosclerosis, or allograft, their roles in the vascular remodeling process of an arterial BVG remains unknown. Thus, we assessed the neottisue formation on arterial BVG remodeling by administrating imatinib, which is both a platelet-derived growth factor receptor kinase inhibitor and c-Kit receptor kinase inhibitor, in a murine model. METHODS: BVGs were composed of an inner poly(L-lactic-co-ε-caprolactone) copolymer sponge layer and an outer electrospun poly(L-lactic acid) nanofiber layer, which were implanted into the infrarenal abdominal aortas of C57BL/6 mice. After graft implantation, saline or 100 mg/kg of imatinib was administrated intraperitoneally daily for 2 weeks (n = 20 per group). Five mice in each group were scheduled to be humanely killed at 3 weeks and 15 at 8 weeks, and BVGs were explanted for histologic assessments. RESULTS: Graft patency during the 8-week observational period was not significantly different between groups (control, 86.7% vs imatinib, 80.0%; P > .999). Neotissue formation consisting of endothelialization, smooth muscle proliferation, and deposition of collagen and elastin was not observed in either group at 3 weeks. Similar endothelialization was achieved in both groups at 8 weeks, but thickness and percent area of neotissue formation were significantly higher in the control group than in the imatinib group, (thickness, 30. 1 ± 7.2 µm vs 19.6 ± 4.5 µm [P = .001]; percent area, 9.8 ± 2.7% vs 6.8 ± 1.8% [P = .005]). Furthermore, SMC layer and deposition of collagen and elastin were better organized at 8 weeks in the control group compared with the imatinib group. The thickness of SMC layer and collagen fiber area were significantly greater at 8 weeks in the control group than in the imatinib group (P < .001 and P = .026, respectively). Because there was no difference in the inner diameter of explanted BVGs (831.7 ± 63.4 µm vs 841.8 ± 41.9 µm; P = .689), neotissue formation was thought to advance toward the outer portion of the BVG with degradation of the polymer scaffold. CONCLUSIONS: Imatinib attenuates neotissue formation during vascular remodeling in arterial bioresorbable vascular grafts (BVGs) by inhibiting SMC layer formation and extracellular matrix deposition. CLINICAL RELEVANCE: This study demonstrated that imatinib attenuated neotissue formation during vascular remodeling in arterial Bioresorbable vascular graft (BVG) by inhibiting smooth muscle cell formation and extracellular matrix deposition. In addition, as imatinib did not modify the inner diameter of BVG, neotissue advanced circumferentially toward the outer portion of the neovessel. Currently, BVGs have not yet been clinically applied to the arterial circulation. The results of this study are helpful for the design of BVG that can achieve an optimal balance between polymer degradation and neotissue formation.

In vivo implantation of 3-dimensional printed customized branched tissue engineered vascular graft in a porcine model.

BACKGROUND: The customized vascular graft offers the potential to simplify the surgical procedure, optimize physiological function, and reduce morbidity and mortality. This experiment evaluated the feasibility of a flow dynamic-optimized branched tissue engineered vascular graft (TEVG) customized based on medical imaging and manufactured by 3-dimensional (3D) printing for a porcine model. METHODS: We acquired magnetic resonance angiography and 4-dimensional flow data for the native anatomy of the pigs (n = 2) to design a custom-made branched vascular graft of the pulmonary bifurcation. An optimal shape of the branched vascular graft was designed using a computer-aided design system informed by computational flow dynamics analysis. We manufactured and implanted the graft for pulmonary artery (PA) reconstruction in the porcine model. The graft was explanted at 4 weeks after implantation for further evaluation. RESULTS: The custom-made branched PA graft had a wall shear stress and pressure drop (PD) from the main PA to the branch PA comparable to the native vessel. At the end point, magnetic resonance imaging revealed comparable left/right pulmonary blood flow balance. PD from main PA to branch between before and after the graft implantation was unchanged. Immunohistochemistry showed evidence of endothelization and smooth muscle layer formation without calcification of the graft. CONCLUSIONS: Our animal model demonstrates the feasibility of designing and implanting image-guided, 3D-printed, customized grafts. These grafts can be designed to optimize both anatomic fit and hemodynamic properties. This study demonstrates the tremendous potential structural and physiological advantages of customized TEVGs in cardiac surgery.

Early natural history of neotissue formation in tissue-engineered vascular grafts in a murine model.

Aim: To characterize early events in neotissue formation during the first 2 weeks after vascular scaffold implantation. Materials & methods: Biodegradable polymeric scaffolds were implanted as abdominal inferior vena cava interposition grafts in wild-type mice. Results: All scaffolds explanted at day 1 contained a platelet-rich mural thrombus. Within the first few days, the majority of cell infiltration appeared to be from myeloid cells at the peritoneal surface with modest infiltration along the lumen. Host reaction to the graft was distinct between the scaffold and mural thrombus; the scaffold stimulated an escalating foreign body reaction, whereas the thrombus was quickly remodeled into collagen-rich neotissue. Conclusion: Mural thrombi remodel into neotissue that persistently occludes the lumen of vascular grafts.

Intravascular Ultrasound Characterization of a Tissue-Engineered Vascular Graft in an Ovine Model.

Patients who undergo implantation of a tissue-engineered vascular graft (TEVG) for congenital cardiac anomalies are monitored with echocardiography, followed by magnetic resonance imaging or angiography when indicated. While these methods provide data regarding the lumen, minimal information regarding neotissue formation is obtained. Intravascular ultrasound (IVUS) has previously been used in a variety of conditions to evaluate the vessel wall. The purpose of this study was to evaluate the utility of IVUS for evaluation of TEVGs in our ovine model. Eight sheep underwent implantation of TEVGs either unseeded or seeded with bone marrow-derived mononuclear cells. Angiography, IVUS, and histology were directly compared. Endothelium, tunica media, and graft were identifiable on IVUS and histology at multiple time points. There was strong agreement between IVUS and angiography for evaluation of luminal diameter. IVUS offers a valuable tool to evaluate the changes within TEVGs, and clinical translation of this application is warranted.

The role of myeloid cell-derived PDGF-B in neotissue formation in a tissue-engineered vascular graft.

AIM: Inflammatory myeloid lineage cells mediate neotissue formation in tissue-engineered vascular grafts, but the molecular mechanism is not completely understood. We examined the role of vasculogenic PDGF-B in tissue-engineered vascular graft neotissue development. MATERIALS & METHODS: Myeloid cell-specific PDGF-B knockout mice (PDGF-KO) were generated using bone marrow transplantation, and scaffolds were implanted as inferior vena cava interposition grafts in either PDGF-KO or wild-type mice. RESULTS: After 2 weeks, grafts from PDGF-KO mice had more remaining scaffold polymer and less intimal neotissue development. Increased macrophage apoptosis, decreased smooth muscle cell proliferation and decreased collagen content was also observed. CONCLUSION: Myeloid cell-derived PDGF contributes to vascular neotissue formation by regulating macrophage apoptosis, smooth muscle cell proliferation and extracellular matrix deposition.

Osteogenic potential of murine periosteum for critical-size cranial defects.

Tissue engineering of bone has combined bespoke scaffolds and osteoinductive factors to maintain functional osteoprogenitor cells, and the periosteum has been confirmed as a satisfactory source of osteoblasts. Suitable matrices have been identified that support cell proliferation and differentiation, including demineralised bone matrix (both compatible and osteoinductive) and acellular human dermis. We have evaluated the osteogenic potential of an osteogenic unit, developed by combining periosteum, demineralised bone matrix, and acellular human dermis, in rodents with critical-size cranial defects. Briefly, remnants from the superior maxillary periosteum were used to harvest cells, which were characterised by flow cytometry and reverse retrotranscriptase-polymerase chain reaction (RT-PCR). Cells were cultured into the osteogenic unit and assessed for viability before being implanted into 3 rodents, These were compared with the control group (n=3) after three months. Histological analyses were made after staining with haematoxylin and eosin and Von Kossa, and immunostaining, and confirmed viable cells that stained for CD90, CD73, CD166, runt-related transcription factor, osteopontin, and collagen type I in the experimental group, while in the control group there was only connective tissue on the edges of the bone in the injury zone. We conclude that osteogenic unit constructs have the osteogenic and regenerative potential for use in engineering bone tissue.

Evaluation of the Early In Vivo Response of a Functionally Graded Macroporous Scaffold in an Osteochondral Defect in a Rabbit Model.

Cartilage tissue engineering is a multifactorial problem requiring a wide range of material property requirements from provision of biological cues to facilitation of mechanical support in load-bearing diarthrodial joints. The study aim was to design, fabricate and characterize a template to promote endogenous cell recruitment for enhanced cartilage repair. A polylactic acid poly-ε-caprolactone (PLCL) support structure was fabricated using laser micromachining technology and thermal crimping to create a functionally-graded open pore network scaffold with a compressive modulus of 9.98 ± 1.41 MPa and a compressive stress at 50% strain of 8.59 ± 1.35 MPa. In parallel, rabbit mesenchymal stem cells were isolated and their growth characteristics, morphology and multipotency confirmed. Sterilization had no effect on construct chemical structure and cellular compatibility was confirmed. After four weeks implantation in an osteochondral defect in a rabbit model to assess biocompatibility, there was no evidence of inflammation or giant cells. Moreover, acellular constructs performed better than cell-seeded constructs with endogenous progenitor cells homing through microtunnels, differentiating to form neo-cartilage and strengthening integration with native tissue. These results suggest, albeit at an early stage of repair, that by modulating the architecture of a macroporous scaffold, pre-seeding with MSCs is not necessary for hyaline cartilage repair.

Coupling curvature-dependent and shear stress-stimulated neotissue growth in dynamic bioreactor cultures: a 3D computational model of a complete scaffold.

The main challenge in tissue engineering consists in understanding and controlling the growth process of in vitro cultured neotissues toward obtaining functional tissues. Computational models can provide crucial information on appropriate bioreactor and scaffold design but also on the bioprocess environment and culture conditions. In this study, the development of a 3D model using the level set method to capture the growth of a microporous neotissue domain in a dynamic culture environment (perfusion bioreactor) was pursued. In our model, neotissue growth velocity was influenced by scaffold geometry as well as by flow- induced shear stresses. The neotissue was modeled as a homogenous porous medium with a given permeability, and the Brinkman equation was used to calculate the flow profile in both neotissue and void space. Neotissue growth was modeled until the scaffold void volume was filled, thus capturing already established experimental observations, in particular the differences between scaffold filling under different flow regimes. This tool is envisaged as a scaffold shape and bioprocess optimization tool with predictive capacities. It will allow controlling fluid flow during long-term culture, whereby neotissue growth alters flow patterns, in order to provide shear stress profiles and magnitudes across the whole scaffold volume influencing, in turn, the neotissue growth.

Synoviocyte neotissues towards in vitro meniscal tissue engineering.

Meniscal injuries are a common cause of pain and osteoarthritis in dogs. We describe here the production of synoviocyte-derived autologous neotissues for potential application in meniscal tissue engineering, via two different culture techniques: contracted or tensioned synthesis of synoviocyte neotissues. Synoviocytes were obtained during routine stifle arthroscopy and cultured from 14 dogs with naturally occurring osteoarthritis of the stifle. Neotissues were analyzed for meniscal-like matrix components and their gene expression, inflammatory gene expression, and cell viability. Tension improved cell viability, and, independent of cell viability, fibrochondrogenic activity by promoting expression of collagen type 1 and aggrecan genes and attenuating gene expression of IL-6. Through this mechanism tension increased collagen protein content and chondrogenic index of neotissues. Alpha smooth muscle actin was present in all neotissues and was responsible for grossly visible contractile behavior. Application of tension to synoviocytes may be a viable culture method towards in vitro meniscal tissue engineering.