Form and functional repair of long bone using 3D‐printed bioactive scaffolds

Injuries to the extremities often require resection of necrotic hard tissue. For large‐bone defects, autogenous bone grafting is ideal but, similar to all grafting procedures, is subject to limitations. Synthetic biomaterial‐driven engineered healing offers an alternative approach. This work focuses on three‐dimensional (3D) printing technology of solid‐free form fabrication, more specifically robocasting/direct write. The research hypothesizes that a bioactive calcium‐phosphate scaffold may successfully regenerate extensive bony defects in vivo and that newly regenerated bone will demonstrate mechanical properties similar to native bone as healing time elapses. Robocasting technology was used in designing and printing customizable scaffolds, composed of 100% beta tri‐calcium phosphate (β‐TCP), which were used to repair critical sized long‐bone defects. Following full thickness segmental defects (~11 mm × full thickness) in the radial diaphysis in New Zealand white rabbits, a custom 3D‐printed, 100% β‐TCP, scaffold was implanted or left empty (negative control) and allowed to heal over 8, 12, and 24 weeks. Scaffolds and bone, en bloc, were subjected to micro‐CT and histological analysis for quantification of bone, scaffold and soft tissue expressed as a function of volume percentage. Additionally, biomechanical testing at two different regions, (a) bone in the scaffold and (b) in native radial bone (control), was conducted to assess the newly regenerated bone for reduced elastic modulus (E_r) and hardness (H) using nanoindentation. Histological analysis showed no signs of any adverse immune response while revealing progressive remodelling of bone within the scaffold along with gradual decrease in 3D‐scaffold volume over time. Micro‐CT images indicated directional bone ingrowth, with an increase in bone formation over time. Reduced elastic modulus (E_r) data for the newly regenerated bone presented statistically homogenous values analogous to native bone at the three time points, whereas hardness (H) values were equivalent to the native radial bone only at 24 weeks. The negative control samples showed limited healing at 8 weeks. Custom engineered β‐TCP scaffolds are biocompatible, resorbable, and can directionally regenerate and remodel bone in a segmental long‐bone defect in a rabbit model. Custom designs and fabrication of β‐TCP scaffolds for use in other bone defect models warrant further investigation.     

Evaluation of in vitro spermatogenesis using poly(D,L‐lactic‐co‐glycolic acid) (PLGA)‐based macroporous biodegradable scaffolds

Successful in vitro differentiation of spermatogenic cells into spermatids appears to offer extremely attractive potential for the treatment of impaired spermatogenesis and male infertility. Experimental evidence indicates that biocompatible polymers may improve in vitro reconstitution and regeneration of tissues of various origins. Here, we fabricated highly porous biodegradable poly(D,L ‐lactic‐co‐glycolic acid) or PLGA co‐polymer scaffolds by combining the gas‐foaming and salt‐leaching methods, using ammonium bicarbonate as a porogen, which allowed us to generate polymer scaffolds with a high density of interconnected pores of 400–500 µm in average diameter, concomitant with a high malleability to mould a wide range of temporal tissue scaffolds requiring a specific shape and geometry. The PLGA scaffolds were biocompatible and biodegradable, as evidenced by the fact that they survived almost 3 month long subcutaneous xenografting into immunodeficient host mice and became easily destroyable after recovery. Immature rat testicular cells that were seeded onto the surface of the scaffold exhibited about 65% seeding efficiency and up to 75% viability after 18 days in culture. Furthermore, our scaffolds enhanced the proliferation and differentiation of spermatogenic germ cells to a greater extent than conventional in vitro culture methods, such as monolayer or organ culture. Taken together, an implication of the present findings is that the PLGA‐based macroporous scaffold may provide a novel means by which spermatocytes could be induced to differentiate into presumptive spermatids. Copyright © 2010 John Wiley & Sons, Ltd.     

A collagen cardiac patch incorporating alginate microparticles permits the controlled release of hepatocyte growth factor and insulin‐like growth factor‐1 to enhance cardiac stem cell migration and proliferation

Cardiac stem cells (CSCs) represent a logical cell type to exploit as a regenerative treatment option for tissue damage accrued as a result of a myocardial infarction. However, the isolation and expansion of CSCs prior to cell transplantation is time consuming, costly and invasive, and the reliability of cell expansion may also prove to be a major obstacle in the clinical application of CSC‐based transplantation therapy after a myocardial infarction. In order to overcome this, we propose the incorporation of growth factor‐eluting alginate microparticles into collagen‐based scaffolds as an implantable biomaterial to promote the recruitment and expansion of CSCs in the myocardium. In order to obtain scaffolds able to enhance the motogenic and proliferative potential of CSCs, the aim of this work was to achieve a sustained delivery of both hepatocyte growth factor and insulin‐like growth factor‐1. Both proteins were initially encapsulated in alginate microparticles by spray drying and subsequently incorporated into a collagen scaffold. Microparticles were seen to homogeneously distribute through the interconnected scaffold pore structure. The resulting scaffolds were capable of extending the release of both proteins up to 15 days, a three‐fold increase over non‐encapsulated proteins embedded in the scaffolds. In vitro assays with isolated CSCs demonstrated that the sustained release of both bioactive proteins resulted in an increased motogenic and proliferative effect. As presently practiced, the isolation and expansion of CSCs for autologous cell transplantation is slow, expensive and difficult to attain. Thus, there is a need for strategies to specifically activate in situ the intrinsic cardiac regenerative potential represented by the CSCs using combinations of growth factors obviating the need for cell transplantation. By favouring the natural regenerative capability of CSCs, it is hypothesized that the cardiac patch presented here will result in positive therapeutic outcomes in MI and heart failure patients in the future. Copyright © 2016 John Wiley & Sons, Ltd.     

Surface modification of titanium with hydroxyapatite–heparin–BMP‐2 enhances the efficacy of bone formation and osseointegration in vitro and in vivo

Surface‐modified titanium (Ti) samples with hydroxyapatite (HAp) and heparin (Hep)–bone morphogenetic protein‐2 (BMP‐2) complex (Ti/HAp/Hep/BMP‐2) were prepared, and their efficacies on the enhancements of bone formation and osseointegration in vitro and in vivo were examined as compared to Ti/HAp and Ti/Hep/BMP‐2. The modified surfaces were characterized by X‐ray photoelectron spectroscopy (XPS), scanning electron microscopy (SEM) and contact angle goniometry. In vitro studies revealed that MG‐63 human osteosarcoma cell lines grown on Ti/HAp/Hep/BMP‐2 increased the amounts of alkaline phosphatase (ALP) activity, calcium deposition and the levels of OCN mRNA gene expression as compared to those grown on Ti/HAp, Ti/Hep/BMP‐2 or pristine Ti. Moreover, Ti/HAp/Hep/BMP‐2 exhibited higher bone volume (BV), bone volume/tissue volume (BV/TV), removal torque value and bone–implant contact (BIC) than Ti/HAp, Ti/Hep/BMP‐2 or pristine Ti in vivo. Histological evaluations showed that many desirable features of bone remodelling existed at the interface between Ti/HAp/Hep/BMP‐2 and the host bone. Consequently, Ti/HAp/Hep/BMP‐2 may have potential for clinical use as dental or orthopaedic implants. Copyright © 2014 John Wiley & Sons, Ltd.     

Local delivery of allogeneic bone marrow and adipose tissue‐derived mesenchymal stromal cells for cutaneous wound healing in a porcine model

Wound healing remains a major challenge in modern medicine. Bone marrow‐ (BM) and adipose tissue‐ (AT) derived mesenchymal stromal/stem cells (MSCs) are of great interest for tissue reconstruction due to their unique immunological properties and regenerative potential. The purpose of this study was to characterize BM and AT‐MSCs and evaluate their effect when administered in a porcine wound model. MSCs were derived from male Göttingen Minipigs and characterized according to established criteria. Allogeneic BM‐ or AT‐MSCs were administered intradermally (1 x 10⁶ cells) into partial‐thickness wounds created on female animals, and covered with Vaseline® gauze or fibrin in a randomized pattern. Animals were euthanized at 7, 10, 14 and 21 days. Tissues were analyzed visually for healing and by microscopic examination for epidermal development and remodelling. Polymerase chain reaction (PCR) was used to detect the presence of male DNA in the specimens. All wounds were healed by 14 days. MSC‐injected wounds were associated with improved appearance and faster re‐epithelialization compared to saline controls. Evaluation of rete ridge depth and architecture showed that MSC treatment promoted a faster rate of epidermal maturation. Male DNA was detected in all samples at days 7 and 10, suggesting the presence of MSCs. We showed the safety, feasibility and potential efficacy of local injection of allogeneic BM‐ and AT‐MSCs for treatment of wounds in a preclinical model. Our data in this large animal model support the potential use of BM‐ and AT‐MSC for treatment of cutaneous wounds through modulation of healing and epithelialization. Copyright © 2013 John Wiley & Sons, Ltd.     

Decellularization of bovine small intestinal submucosa and its use for the healing of a critical‐sized full‐thickness skin defect,alone and in combination with stem cells,in a small rodent model

In this study, we initially described an efficient decellularization protocol for bovine‐derived small intestinal submucosa (bSIS), involving freeze–thaw cycles, acid/base treatment and alcohol and buffer systems. We compared the efficacy of our protocol to some previously established ones, based on DNA content and SEM and histochemical analyses. DNA content was reduced by ~89.4%, significantly higher than compared protocols. The sulphated GAG content of the remaining interconnected fibrous structure was 5.738 ± 0.207 µg/mg (55% retained). An in vitro study was performed to evaluate whether rat bone marrow mesenchymal stem cells (MSCs) could attach and survive on bSIS membranes. Our findings revealed that MSCs can preserve their viability and proliferate on bSIS for > 2 weeks in culture. We conducted in vivo applications for the treatment of an experimental rat model of critical sized (7 cm²) full‐thickness skin defect. The wound models treated with either MSCs‐seeded (1.5 × 10⁶ cells/cm²) or non‐seeded bSIS membranes were completely closed by week 7 without significant differences in closure time; on the other hand, the open wound control was closed at ~47% at this time point. Immunohistopathology results revealed that the group which received MSCs‐seeded bSIS had less scarring at the end of the healing process and was in further stages of appendage formation in comparison with the non‐seeded bSIS group. Copyright © 2015 John Wiley & Sons, Ltd.     

Angiogenic and osteogenic regeneration in rats via calcium phosphate scaffold and endothelial cell co‐culture with human bone marrow mesenchymal stem cells (MSCs), human umbilical cord MSCs,human induced pluripotent stem cell‐derived MSCs and human embryonic stem cell‐derived MSCs

Angiogenesis is a limiting factor in regenerating large bone defects. The objective of this study was to investigate angiogenic and osteogenic effects of co‐culture on calcium phosphate cement (CPC) scaffold using human umbilical vein endothelial cells (hUVECs) and mesenchymal stem cells (MSCs) from different origins for the first time. hUVECs were co‐cultured with four types of cell: human umbilical cord MSCs (hUCMSCs), human bone marrow MSCs (hBMSCs) and MSCs from induced pluripotent stem cells (hiPSC‐MSCs) and embryonic stem cells (hESC‐MSCs). Constructs were implanted in 8 mm cranial defects of rats for 12 weeks. CPC without cells served as control 1. CPC with hBMSCs served as control 2. Microcapillary‐like structures were successfully formed on CPC in vitro in all four co‐cultured groups. Microcapillary lengths increased with time (p < 0.05). Osteogenic and angiogenic gene expressions were highly elevated and mineralization by co‐cultured cells increased with time (p < 0.05). New bone amount and blood vessel density of co‐cultured groups were much greater than controls (p < 0.05) in an animal study. hUVECs co‐cultured with hUCMSCs, hiPSC‐MSCs and hESC‐MSCs achieved new bone and vessel density similar to hUVECs co‐cultured with hBMSCs (p > 0.1). Therefore, hUCMSCs, hiPSC‐MSCs and hESC‐MSCs could serve as alternative cell sources to hBMSCs, which require an invasive procedure to harvest. In conclusion, this study showed for the first time that co‐cultures of hUVECs with hUCMSCs, hiPSC‐MSCs, hESC‐MSCs and hBMSCs delivered via CPC scaffold achieved excellent osteogenic and angiogenic capabilities in vivo. The novel co‐culture constructs are promising for bone reconstruction with improved angiogenesis for craniofacial/orthopaedic applications. Copyright © 2017 John Wiley & Sons, Ltd.     

Using minimalist self‐assembling peptides as hierarchical scaffolds to stabilise growth factors and promote stem cell integration in the injured brain

Neurotrophic growth factors are effective in slowing progressive degeneration and/or promoting neural repair through the support of residual host and/or transplanted neurons. However, limitations including short half‐life and enzyme susceptibility of growth factors highlight the need for alternative strategies to prolong localised delivery at a site of injury. Here, we establish the utility of minimalist N‐fluorenylmethyloxycarbonyl (Fmoc) self‐assembling peptides (SAPs) as growth factor delivery vehicle, targeted at supporting neural transplants in an animal model of Parkinson's disease. The neural tissue‐specific SAP, Fmoc‐DIKVAV, demonstrated sustained release of glial cell line derived neurotrophic factor, up to 172 hr after gel loading. This represents a significant advance in drug delivery, because its lifetime in phosphate buffered saline was less than 1 hr. In vivo transplantation of neural progenitor cells, together with our growth factor‐loaded material, into the injured brain improved graft survival compared with cell transplants alone. We show for the first time the use of minimalist Fmoc‐SAP in an in vivo disease model for sustaining the delivery of neurotrophic growth factors, facilitating their spatial and temporal delivery in vivo, whilst also providing an enhanced niche environment for transplanted cells.     

IDR‐1018 induces cell proliferation,migration, and reparative gene expression in 2D culture and 3D human skin equivalents

Skin lesions are associated with functional/cosmetic problems for those afflicted. Scarless regeneration is a challenge, not limited to the skin, and focus of active investigation. Recently, the host defense peptide innate defense regulatory peptide 1018 (IDR‐1018) has shown exciting regenerative properties. Nevertheless, literature regarding IDR‐1018 regenerative potential is scarce and limited to animal models. Here, we evaluated the regenerative potential of IDR‐1018 using human 2D and 3D human skin equivalents. First, we investigated IDR‐1018 using human cells found in skin—primary fibroblasts, primary keratinocytes, and the MeWo melanocytes cell line. IDR‐1018 promoted cell proliferation and expression of marker of proliferation Ki‐67, matrix metalloproteinase 1, and hyaluronan synthase 2 by fibroblasts. In keratinocytes, a drastic increase in expression was observed for Ki‐67, matrix metalloproteinase 1, C‐X‐C motif chemokine receptor type 4, C‐X‐C motif chemokine receptor type 7, fibroblast growth factor 2, hyaluronan synthase 2, vascular endothelial growth factor, and elastin, reflecting an intense stimulation of these cells. In melanocytes, increased migration and proliferation were observed following IDR‐1018 treatment. The capacity of IDR‐1018 to promote dermal contraction was verified using a dermal model. Finally, using a 3D human skin equivalent lesion model, we revealed that the regenerative potential of IDR1018, previously tested in mice and pigs, is valid for human skin tissue. Lesions closed faster in IDR‐1018‐treated samples, and the gene expression signature observed in 2D was reproduced in the 3D human skin equivalents. Overall, the present data show the regenerative potential of IDR‐1018 in an experimental system comprising human cells, underscoring the potential application for clinical investigation.     

Characterization of a human fetal spinal cord stem cell line,NSI‐566RSC,and its induction to functional motoneurons

Specific neuronal subtypes, especially motoneurons (MNs), derived from human stem cells provide a significant therapeutic potential for spinal cord diseases, such as amyotrophic lateral sclerosis (ALS) and spinal cord injury. So far, in vitro, MNs have only been successfully induced from embryonic stem cells (hESC) and human fetal cortical progenitors. Although neural progenitors from spinal cord would be a likely source for generating MNs, there has been no study reporting successful in vitro differentiation of MNs from spinal cord progenitors. This study first characterized a polyclonal spinal cord stem cell line isolated from an 8 week‐old fetus. Then a paradigm was introduced to successfully induce MNs from this cell line, which was demonstrated by immunostaining using the MN markers HB9, Islet1 and choline acetyl transferase (ChAT). The combination of HB9 and ChAT immunostainings indicated that ～20% of the cells were MNs after this induction protocol. The presence of other cell types in the differentiated culture was also analysed. Finally, the electrophysiological properties of these differentiated MNs were characterized to confirm their functional integrity. The majority of these MNs fired repetitive action potentials (APs), which is an indicator of functional maturation. The recordings of spontaneous excitatory postsynaptic currents (EPSCs) confirmed the formation of synapses onto these MNs. This study reports the first successful differentiation of MNs from human spinal cord stem cells in vitro, providing a novel approach for obtaining functional MNs when designing the therapeutic strategy for spinal cord diseases or injuries. Copyright © 2009 John Wiley & Sons, Ltd.