Bioactive copolymer scaffolds for bone tissue engineering. Efficacy and host response

Current research focuses on developing a novel bone-inducing scaffold that could deliver controlled osteogenic growth factors. Several aspects, in particular those influencing the efficacy of such bioactive scaffolds, such as release kinetics of the growth factor, biocompatibility and biodegradability, need further study. The aim of this thesis was to determine a mode of bone morphogenetic protein-2 (BMP-2) delivery from copolymer scaffolds that reduce the dose to improve clinical safety while retaining efficacy. A low dose of 1 μg BMP-2 was immobilised via four different functionalising techniques on recently developed poly(LLA-co-CL) scaffolds. Sustained release of low levels was seen from BMP-2 physisorbed on nanodiamond modified scaffolds (nDP-PHY) for up to 70 days in vitro compared to that from scaffolds modified with microspheres containing BMP-2 (MICS) and unmodified scaffolds with physisorbed BMP-2 (PHY). No release was detected from BMP-2 covalently bound to nanodiamond modified scaffolds. nDP-PHY, MICS and PHY scaffolds promoted bone regeneration in a rat mandible critical-sized defect after 4 weeks, however, nDP-PHY and MICS scaffolds demonstrated osteogenic potential in vivo as well as in mesenchymal stem/stromal cell (MSC) cultures. Poly(LLA-co-CL) scaffolds modified with nanodiamond (nDP) and nDP with physisorbed BMP-2 were then evaluated through in vivo degradation, host tissue response and tumorigenic potential. Modified scaffolds degraded faster than unmodified scaffolds. Gene expression of proinflammatory, osteogenic and angiogenic markers were upregulated in the nDP and nDP-PHY scaffolds with ectopic bone seen at week 8 only from the latter. Inflammatory cells, foreign body giant cells and fibrous capsule tissue were significantly reduced around the modified scaffolds. Tissue regeneration markers were most highly expressed in the modified groups. Interestingly, nanodiamond particles were found in the implantation site after 27 weeks when 90% of the scaffolds had degraded. To evaluate the tumorigenic potential of the functionalised scaffolds in vivo, a sensitive and non-invasive model using xenotransplantation of early neoplastic oral keratinocytes transfected to express luciferase (DOKLuc) together with carcinoma associated fibroblasts (CAF) for monitoring microenvironmentally-induced carcinogenesis was developed. nDP scaffolds without BMP-2 reduced the bioluminescence intensity of positive control tumours formed by DOKLuc+CAF in vivo. When cultured in vitro as 3D organotypic models of neoplastic oral mucosa, DOKLuc previously cultured on nDP scaffolds demonstrated reduced tumorigenic potential compared to DOKLuc from nDP-PHY and unmodified scaffolds. nDP-PHY scaffolds showed enhanced tumorigenic potential in vivo and in vitro. These results suggest a role played by nanodiamonds in reducing tumorigenic potential of DOKLuc and also raises concerns for the therapeutic use of BMP-2 for the reconstruction of bone defects in oral cancer patients. This thesis also highlights that the mode of binding BMP-2 to a scaffold has a significant effect on its osteogenic potential. Furthermore, the efficacy of delivering low, sustained amounts of BMP-2 is emphasised and the modality of nDP-PHY is shown to provide a promising bioactive scaffold for bone tissue engineering

Bioactive copolymer scaffolds for bone tissue engineering. Efficacy and host response

Current research focuses on developing a novel bone-inducing scaffold that could deliver controlled osteogenic growth factors. Several aspects, in particular those influencing the efficacy of such bioactive scaffolds, such as release kinetics of the growth factor, biocompatibility and biodegradability, need further study. The aim of this thesis was to determine a mode of bone morphogenetic protein-2 (BMP-2) delivery from copolymer scaffolds that reduce the dose to improve clinical safety while retaining efficacy. A low dose of 1 μg BMP-2 was immobilised via four different functionalising techniques on recently developed poly(LLA-co-CL) scaffolds. Sustained release of low levels was seen from BMP-2 physisorbed on nanodiamond modified scaffolds (nDP-PHY) for up to 70 days in vitro compared to that from scaffolds modified with microspheres containing BMP-2 (MICS) and unmodified scaffolds with physisorbed BMP-2 (PHY). No release was detected from BMP-2 covalently bound to nanodiamond modified scaffolds. nDP-PHY, MICS and PHY scaffolds promoted bone regeneration in a rat mandible critical-sized defect after 4 weeks, however, nDP-PHY and MICS scaffolds demonstrated osteogenic potential in vivo as well as in mesenchymal stem/stromal cell (MSC) cultures. Poly(LLA-co-CL) scaffolds modified with nanodiamond (nDP) and nDP with physisorbed BMP-2 were then evaluated through in vivo degradation, host tissue response and tumorigenic potential. Modified scaffolds degraded faster than unmodified scaffolds. Gene expression of proinflammatory, osteogenic and angiogenic markers were upregulated in the nDP and nDP-PHY scaffolds with ectopic bone seen at week 8 only from the latter. Inflammatory cells, foreign body giant cells and fibrous capsule tissue were significantly reduced around the modified scaffolds. Tissue regeneration markers were most highly expressed in the modified groups. Interestingly, nanodiamond particles were found in the implantation site after 27 weeks when 90% of the scaffolds had degraded. To evaluate the tumorigenic potential of the functionalised scaffolds in vivo, a sensitive and non-invasive model using xenotransplantation of early neoplastic oral keratinocytes transfected to express luciferase (DOKLuc) together with carcinoma associated fibroblasts (CAF) for monitoring microenvironmentally-induced carcinogenesis was developed. nDP scaffolds without BMP-2 reduced the bioluminescence intensity of positive control tumours formed by DOKLuc+CAF in vivo. When cultured in vitro as 3D organotypic models of neoplastic oral mucosa, DOKLuc previously cultured on nDP scaffolds demonstrated reduced tumorigenic potential compared to DOKLuc from nDP-PHY and unmodified scaffolds. nDP-PHY scaffolds showed enhanced tumorigenic potential in vivo and in vitro. These results suggest a role played by nanodiamonds in reducing tumorigenic potential of DOKLuc and also raises concerns for the therapeutic use of BMP-2 for the reconstruction of bone defects in oral cancer patients. This thesis also highlights that the mode of binding BMP-2 to a scaffold has a significant effect on its osteogenic potential. Furthermore, the efficacy of delivering low, sustained amounts of BMP-2 is emphasised and the modality of nDP-PHY is shown to provide a promising bioactive scaffold for bone tissue engineering

Pre-admission Grades And Student Performance: The Malaysian Medical School Experience

Prior academic achievement is often considered the best predictor and therefore the preadmission criteria for highly competitive medical schools. Most studies that advocate this viewpoint analyzed cohorts based on results of a central examination taken by students who come from various pre-university setups and backgrounds. Far less is known about students who come from a common setup, sit for common assessments, and the effect on their achievement in medical schools. This study sets out to investigate the correlation and association of preadmission grades and various summative results during preclinical year medical program. The association of the whole group is investigated. The association of subgroups (low, intermediate, and high achievers) to preclinical achievement was also investigated to get insight about the consistency. As a group, the pre-university performance (especially in natural sciences) has moderate to high correlations to various results in preclinical phase. As subgroups: low achievers are less consistent and predictable; high achievers are best correlated to various results of preclinical phase; while intermediate achievers are in between. The cumulative grade point and performance in natural sciences in a common pre-university program can serve not only as a predictor of performance, but also as an indicator for consistency of performance in preclinical phase of medical programs. This information may be of use to medical school admission and selection committees

Extracellular Vesicles Derived from Primed Mesenchymal Stromal Cells Loaded on Biphasic Calcium Phosphate Biomaterial Exhibit Enhanced Macrophage Polarization

Mesenchymal stromal cells (MSC) loaded on biphasic calcium phosphate biomaterial (MSC + BCP) have been used as an advanced therapy medicinal product to treat complex maxillofacial bone defects in patients. Further, MSC-derived extracellular vesicles (EVs) are established vehicles of paracrine factors, supporting inter-cellular communication between MSC and other interacting cell types, such as monocytes/macrophages. However, the information about the immunomodulatory potential of EVs derived from MSC and biomaterial constructs (MSC + BCP:EV) and inflammatory primed constructs (MSCp + BCP:EV) are scarce. Hence, we isolated and characterized EVs from these different systems, and compared their cytokine contents with plastic-adherent MSC-derived EVs (MSC:EV). When EVs from all three MSC systems were added to the primary blood-derived macrophages in vitro, significantly higher numbers of M0 (naive) macrophages shifted to M2-like (anti-inflammatory) by MSCp + BCP:EV treatment. Further, this treatment led to enhanced switching of M1 polarized macrophages to M2 polarized, and conversely, M2 to M1, as evaluated by determining the M1/M2 ratios after treatment. The enhanced macrophage modulation by MSCp + BCP:EV was attributed to their higher immunomodulatory (TNFα, IL1β, IL5), angiogenic (VEGF), and chemokine-rich (RANTES, MCP1, MIP1β) cytokine cargo. In conclusion, we successfully isolated and characterized EVs from MSC + BCP constructs and demonstrated that, depending upon the tissue microenvironment, these EVs contribute towards modulating the macrophage-mediated inflammation and healing responses. The study offers new insights into the use of biomaterial-induced EVs for MSC secretome delivery, as a step towards future ‘cell-free’ bone regenerative therapies.publishedVersio

Inflammatory mediators in saliva and gingival fluid of children with congenital heart defect

Objectives (a) To compare levels of pro- and anti-inflammatory mediators in saliva and gingival crevicular fluid (GCF) in children with and without congenital heart defects (CHD cases and controls) and to test whether a systemic component exists in CHD cases by controlling for gingivitis and plaque scores. (b) To correlate the levels of pro- and anti-inflammatory mediators in GCF and saliva with plaque bacterial composition among CHD cases and controls. Materials and Methods Whole un-stimulated saliva and GCF samples were collected (60 CHD cases, 60 controls [Sudan]) and were analysed for levels of prostaglandin E2 (PGE2), interleukin-1β (IL-1β), tumour necrosis factor-α (TNF-α), interleukin-1ra (IL-1ra) and interleukin-10 (IL-10) levels. These levels were correlated with the previously reported levels of four red complex bacteria. Results Significantly elevated levels of PGE2 and IL-1β in GCF and IL-1β and TNF-α in saliva were detected among CHD cases compared with controls. General linear model (GLM) analyses revealed that PGE2 and IL-1β levels remained significantly higher in GCF and saliva samples, respectively, among CHD cases after controlling for gingivitis and plaque score, whereas TNF-α and IL-10 levels were significantly lower in their GCF samples. Additionally, IL-1β level was significantly positively correlated to the counts of the four red complex species in their GCF. Conclusion In addition to higher levels of some pro-inflammatory mediators in saliva and GCF corresponding to more gingivitis in CHD cases, also a systemic inflammatory component exists and is reflected in these two oral fluids.publishedVersio

RvE1 Impacts the Gingival Inflammatory Infiltrate by Inhibiting the T Cell Response in Experimental Periodontitis

Periodontitis is a chronic inflammatory disease associated with the formation of dysbiotic plaque biofilms and characterized by the progressive destruction of the alveolar bone. The transition from health to disease is characterized by a shift in periodontal immune cell composition, from mostly innate (neutrophils) to adaptive (T lymphocytes) immune responses. Resolvin E1 (RvE1) is a specialized pro-resolution mediator (SPMs), produced in response to inflammation, to enhance its resolution. Previous studies have indicated the therapeutic potential of RvE1 in periodontal disease; however, the impact of RvE1 in the microbial-elicited osteoclastogenic immune response remains uncharacterized in vivo. In the present study, we studied the impact of RvE1 on the gingival inflammatory infiltrate formation during periodontitis in a mouse model. First, we characterized the temporal-dependent changes of the main immune cells infiltrating the gingiva by flow cytometry. Then, we evaluated the impact of early or delayed RvE1 administration on the gingival immune infiltration and cervical lymph nodes composition. We observed a consistent inhibitory outcome on T cells -particularly effector T cells- and a protective effect on regulatory T cells (Tregs). Our data further demonstrated the wide range of actions of RvE1, its preventive role in the establishment of the adaptive immune response during inflammation, and bone protective capacity.publishedVersio

Contact osteogenesis by biodegradable 3D-printed poly(lactide-co-trimethylene carbonate)

Background To support bone regeneration, 3D-printed templates function as temporary guides. The preferred materials are synthetic polymers, due to their ease of processing and biological inertness. Poly(lactide-co-trimethylene carbonate) (PLATMC) has good biological compatibility and currently used in soft tissue regeneration. The aim of this study was to evaluate the osteoconductivity of 3D-printed PLATMC templates for bone tissue engineering, in comparison with the widely used 3D-printed polycaprolactone (PCL) templates. Methods The printability and physical properties of 3D-printed templates were assessed, including wettability, tensile properties and the degradation profile. Human bone marrow-derived mesenchymal stem cells (hBMSCs) were used to evaluate osteoconductivity and extracellular matrix secretion in vitro. In addition, 3D-printed templates were implanted in subcutaneous and calvarial bone defect models in rabbits. Results Compared to PCL, PLATMC exhibited greater wettability, strength, degradation, and promoted osteogenic differentiation of hBMSCs, with superior osteoconductivity. However, the higher ALP activity disclosed by PCL group at 7 and 21 days did not dictate better osteoconductivity. This was confirmed in vivo in the calvarial defect model, where PCL disclosed distant osteogenesis, while PLATMC disclosed greater areas of new bone and obvious contact osteogenesis on surface. Conclusions This study shows for the first time the contact osteogenesis formed on a degradable synthetic co-polymer. 3D-printed PLATMC templates disclosed unique contact osteogenesis and significant higher amount of new bone regeneration, thus could be used to advantage in bone tissue engineering.publishedVersio

Bone regeneration in rat calvarial defects using dissociated or spheroid mesenchymal stromal cells in scaffold-hydrogel constructs

Background Three-dimensional (3D) spheroid culture can promote the osteogenic differentiation of bone marrow mesenchymal stromal cells (BMSC). 3D printing offers the possibility to produce customized scaffolds for complex bone defects. The aim of this study was to compare the potential of human BMSC cultured as 2D monolayers or 3D spheroids encapsulated in constructs of 3D-printed poly-L-lactide-co-trimethylene carbonate scaffolds and modified human platelet lysate hydrogels (PLATMC-HPLG) for bone regeneration. Methods PLATMC-HPLG constructs with 2D or 3D BMSC were assessed for osteogenic differentiation based on gene expression and in vitro mineralization. Subsequently, PLATMC-HPLG constructs with 2D or 3D BMSC were implanted in rat calvarial defects for 12 weeks; cell-free constructs served as controls. Bone regeneration was assessed via in vivo computed tomography (CT), ex vivo micro-CT and histology. Results Osteogenic gene expression was significantly enhanced in 3D versus 2D BMSC prior to, but not after, encapsulation in PLATMC-HPLG constructs. A trend for greater in vitro mineralization was observed in constructs with 3D versus 2D BMSC (p > 0.05). In vivo CT revealed comparable bone formation after 4, 8 and 12 weeks in all groups. After 12 weeks, micro-CT revealed substantial regeneration in 2D BMSC (62.47 ± 19.46%), 3D BMSC (51.01 ± 24.43%) and cell-free PLATMC-HPLG constructs (43.20 ± 30.09%) (p > 0.05). A similar trend was observed in the histological analysis. Conclusion Despite a trend for superior in vitro mineralization, constructs with 3D and 2D BMSC performed similarly in vivo. Regardless of monolayer or spheroid cell culture, PLATMC-HPLG constructs represent promising scaffolds for bone tissue engineering applications.publishedVersio

AXL targeting reduces fibrosis development in experimental unilateral ureteral obstruction

The AXL receptor tyrosine kinase (RTK) is involved in partial epithelial-tomesenchymal transition (EMT) and inflammation – both main promoters of renal fibrosis development. The study aim was to investigate the role of AXL inhibition in kidney fibrosis due to unilateral ureteral obstruction (UUO). Eight weeks old male C57BL/6 mice underwent UUO and were treated with oral AXL inhibitor bemcentinib (n = 22), Angiotensin-converting enzyme inhibitor (ACEI, n = 10), ACEI and bemcentinib (n = 10) or vehicle alone (n = 22). Mice were sacrificed after 7 or 15 days and kidney tissues were analyzed by immunohistochemistry (IHC), western blot, ELISA, Sirius Red (SR) staining, and hydroxyproline (Hyp) quantification. RNA was extracted from frozen kidney tissues and sequenced on an Illumina HiSeq4000 platform. After 15 days the ligated bemcentinib-treated kidneys showed less fibrosis compared to the ligated vehicle-treated kidneys in SR analyses and Hyp quantification. Reduced IHC staining for Vimentin (VIM) and alpha smooth muscle actin (aSMA), as well as reduced mRNA abundance of key regulators of fibrosis such as transforming growth factor (Tgfb), matrix metalloproteinase 2 (Mmp2), Smad2, Smad4, myofibroblast activation (Aldh1a2, Crlf1), and EMT (Snai1,2, Twist), in ligated bemcentinib-treated kidneys was compatible with reduced (partial) EMT induction. Furthermore, less F4/80 positive cells, less activity of pathways related to the immune system and lower abundance of MCP1, MCP3, MCP5, and TARC in ligated bemcentinib-treated kidneys was compatible with reduction in inflammatory infiltrates by bemcentinib treatment. The AXL RTK pathway represents a promising target for pharmacologic therapy of kidney fibrosis.publishedVersio

AXL targeting reduces fibrosis development in experimental unilateral ureteral obstruction

The AXL receptor tyrosine kinase (RTK) is involved in partial epithelial-to-mesenchymal transition (EMT) and inflammation - both main promoters of renal fibrosis development. The study aim was to investigate the role of AXL inhibition in kidney fibrosis due to unilateral ureteral obstruction (UUO). Eight weeks old male C57BL/6 mice underwent UUO and were treated with oral AXL inhibitor bemcentinib (n = 22), Angiotensin-converting enzyme inhibitor (ACEI, n = 10), ACEI and bemcentinib (n = 10) or vehicle alone (n = 22). Mice were sacrificed after 7 or 15 days and kidney tissues were analyzed by immunohistochemistry (IHC), western blot, ELISA, Sirius Red (SR) staining, and hydroxyproline (Hyp) quantification. RNA was extracted from frozen kidney tissues and sequenced on an Illumina HiSeq4000 platform. After 15 days the ligated bemcentinib-treated kidneys showed less fibrosis compared to the ligated vehicle-treated kidneys in SR analyses and Hyp quantification. Reduced IHC staining for Vimentin (VIM) and alpha smooth muscle actin (alpha SMA), as well as reduced mRNA abundance of key regulators of fibrosis such as transforming growth factor (Tgf beta), matrix metalloproteinase 2 (Mmp2), Smad2, Smad4, myofibroblast activation (Aldh1a2, Crlf1), and EMT (Snai1,2, Twist), in ligated bemcentinib-treated kidneys was compatible with reduced (partial) EMT induction. Furthermore, less F4/80 positive cells, less activity of pathways related to the immune system and lower abundance of MCP1, MCP3, MCP5, and TARC in ligated bemcentinib-treated kidneys was compatible with reduction in inflammatory infiltrates by bemcentinib treatment. The AXL RTK pathway represents a promising target for pharmacologic therapy of kidney fibrosis.Peer reviewe