| 1. |
- Gurzawska-Comis, Katarzyna, et al.
(författare)
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GUIDED BONE REGENERATION IN OSTEOPOROSIS BY PLANT-DERIVED NANOPARTICLES
- 2023
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Ingår i: Tissue Engineering. Part A. - : MARY ANN LIEBERT, INC. - 1937-3341 .- 1937-335X. ; 29:11-12, s. 576-577
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Background: The repair and treatment of large bone defects in patients with compromised bone metabolism due to ageing and medical conditions such as osteoporosis present often a clinical challenge. Therefore, adjunctive methods to enhance bone healing are needed.Bone tissue engineering with application of nanotechnology allows to construct biomaterials with desired properties being osteoconductive, osteoinductive and osteogenic.Aim / Hypothesis: The aim of our study was to promote bone regeneration using functionalised scaffold with Rhamnogalacturonan-I pectins (RG-I) in vitro and in vivo using aging and osteoporotic rodent models.Material and Methods: The biomaterials were poly(l-lactide-co-ε-caprolactone) scaffolds and the RG-I was from potato. The chemical and physical properties of functionalised biomaterials with RG-I nanoparticles were characterised using confocal and atomic force microscopy. Functionalised scaffolds with RG-I (tested sample) were evaluated in vitro with human osteoblasts from osteoporotic patients and their response was tested using real-time PCR. In vivo evaluation was performed using critical-size calvaria bone defect model in ageing and osteoporotic rat models. Scaffolds were implanted randomly in the calvaria defects of aged female Wistar rats (11-12 months old) and osteoporotic female Wistar rats induced by ovariectomy. The control was scaffold without RG-I. After 2 and 8 weeks, animals were euthanised. Harvested samples were analysed for osteogenic and inflammatory markers using real-time PCR. Bone formation was evaluated radiographically and histologically. The data was analysed using one-way ANOVA.Results: The chemical and physical properties results indicated success of the functionalisation of scaffolds with RG-I. Osteoblasts response suggested osteogenic (upregulation osteopontin, osteocalcin, collagen1, bone sialoprotein) and anti-inflammatory properties (downregulation IL-1, IL-8, TNF-alpha) on the scaffold functionalised with RG-I. The in vivo results in aged and osteoporotic rat calvaria model of early (2 weeks) bone regeneration showed increase of osteogenic markers and decrease of proinflammatory markers and RANKL, compared to control. In osteoporotic rat model at week 2 and 8 and in aged rat model at week 8, the mean percentage of BV / TV (bone volume / tissue volume) in the defect with RG-I scaffold was significantly greater than the defect with control. The histological evaluation in both rat models revealed larger areas of new bone formation in RG-I scaffolds than in control.Conclusion and Clinical implications: In conclusion, the plant-derived nanoparticles significantly increased osteogenic and decreased pro-inflammatory response in vitro and in vivo. These finding may have a crucial impact on bone repair process especially in elderly and osteoporotic patients.
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| 2. |
- Munir, Arooj, et al.
(författare)
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Efficacy of copolymer scaffolds delivering human demineralised dentine matrix for bone regeneration
- 2019
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Ingår i: Journal of Tissue Engineering. - : SAGE PUBLICATIONS INC. - 2041-7314. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- Poly(L-lactide-co-epsilon-caprolactone) scaffolds were functionalised by 10 or 20 mu g/mL of human demineralised dentine matrix. Release kinetics up to 21 days and their osteogenic potential on human bone marrow stromal cells after 7 and 21 days were studied. A total of 390 proteins were identified by mass spectrometry. Bone regeneration proteins showed initial burst of release. Human bone marrow stromal cells were cultured on scaffolds physisorbed with 20 mu g/mL and cultured in basal medium (DDM group) or physisorbed and cultured in osteogenic medium or cultured on non-functionalised scaffolds in osteogenic medium. The human bone marrow stromal cells proliferated less in demineralised dentine matrix group and activated ERK/1/2 after both time points. Cells on DDM group showed highest expression of IL-6 and IL-8 at 7 days and expressed higher collagen type 1 alpha 2, SPP1 and bone morphogenetic protein-2 until 21 days. Extracellular protein revealed higher collagen type 1 and bone morphogenetic protein-2 at 21 days in demineralised dentine matrix group. Cells on DDM group showed signs of mineralisation. The functionalised scaffolds were able to stimulate osteogenic differentiation of human bone marrow stromal cells.
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| 3. |
- Rashad, Ahmad, et al.
(författare)
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Inflammatory responses and tissue reactions to wood-Based nanocellulose scaffolds
- 2019
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Ingår i: Materials science & engineering. C, biomimetic materials, sensors and systems. - : Elsevier BV. - 0928-4931 .- 1873-0191. ; 97, s. 208-221
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Tidskriftsartikel (refereegranskat)abstract
- Two wood-derived cellulose nanofibril (CNF) porous scaffolds were prepared by TEMPO-oxidation and carboxymethylation. The effects of these scaffolds on the production of inflammatory cytokines by human macrophage-like cells (U937) was profiled in vitro after 1 and 3 days and in subcutaneous tissues of rats after 4 and 30 days, using PCR and Multiplex arrays. Tissue culture plates (TCP) and gelatin scaffolds served as controls in vitro and in vivo respectively. After 3 days in vitro, there was no significant difference between the effects of CNF scaffolds and TCP on the production of chemokines/growth factors and pro-inflammatory cytokines. At day 4 in vivo there was significantly higher gene expression of the anti-inflammatory IL-1Ra in the CNF scaffolds than the gelatin scaffold. Production of IL-1β, IL-6, MCP-1, MIP-1α CXCL-1 and M-CSF was significantly less than in the gelatin, demonstrating an early mild inflammatory response. At day 30, both CNF scaffolds significantly stimulated the production of the anti-inflammatory cytokine IL-10. Unlike gelatin, neither CNF scaffold had degraded 180 days post-implantation. The slow degradation of CNF scaffolds resulted in a foreign body reaction, with high production of IL-1β, IL-2, TNF-α, IFN-ϒ, MCP-1, MIP-1α, M-CSF, VEGF cytokines and expression of MMP-9 gene. The surface chemistry of the CNF scaffolds elicited a modest effect on cytokine production and did not shift the inflammatory profile in vitro or in vivo. The decisive role in development of the foreign body reaction was the slow degradation of the CNF scaffolds.
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| 4. |
- Shanbhag, Siddharth, et al.
(författare)
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Cell therapy for orofacial bone regeneration : A systematic review and meta-analysis
- 2019
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Ingår i: Journal of Clinical Periodontology. - : John Wiley & Sons. - 0303-6979 .- 1600-051X. ; 46:S21, s. 162-182
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Tidskriftsartikel (refereegranskat)abstract
- AIM: The objective of the present review was to answer the focused question: what is the effect of cell therapy in terms of orofacial bone regeneration compared to grafting with only biomaterial scaffolds and/or autogenous bone? METHODS: Electronic databases were searched for relevant controlled clinical and pre-clinical (large-animal) studies. Separate meta-analyses of quantitative data regarding histological or radiographic new bone formation were performed. RESULTS: Forty-seven eligible clinical and 57 pre-clinical studies were included. Clinical studies were categorized based on the use of "minimally manipulated" whole tissues (e.g., bone marrow) or ex vivo expanded cells from "uncommitted" (bone marrow, adipose tissue) or "committed" sources (periosteum, bone). Based on limited and heterogeneous clinical evidence, implantation of cells (mostly whole bone marrow), in combination with biomaterial scaffolds results in bone regeneration which is (a) superior compared to implantation of scaffolds alone in sinus and horizontal ridge augmentation, and (b) comparable to autogenous bone in alveolar cleft repair. CONCLUSIONS: Although current evidence points to the benefits of cell therapy in certain clinical indications, it is unclear whether the use of ex vivo expanded cells, either uncommitted or committed, is superior to whole tissue fractions in terms of bone regeneration. The relatively larger effect sizes in favour of cell therapy observed in pre-clinical studies are diminished in clinical trials. Future controlled studies should include cost-effectiveness analyses to guide clinical decision-making.
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| 5. |
- Shanbhag, Siddharth, et al.
(författare)
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Characterization of Human Gingiva- and PDL-derived Progenitor Cells in Xeno-free Conditions
- 2018
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Objectives: Periodontal tissues represent a clinically relevant and less invasive source of progenitor cells compared to bone marrow, for periodontal- and/or alveolar bone-tissue engineering (P-/BTE). The safety and efficacy of clinical-grade stem cells can be enhanced via substitution of xenogeneic supplements and three-dimensional (3-D) cell culture, respectively, to simulate the in vivo microenvironment more closely. The objective of this study was to comprehensively characterize progenitor cells derived from human gingiva (GPCs) and periodontal-ligament (PDL; PPCs) in xeno-free conditions for use in P-/BTE. Methods: In preliminary experiments, pooled human platelet lysate (PL) was identified as the optimal xeno-substitute for fetal bovine serum (FBS) in stem cell cultures. Primary GPCs and PPCs were isolated and expanded as monolayers in PL- and FBS-supplemented media; passage 3-5 cells were used in experiments. Bone marrow mesenchymal stem cells (BMSCs) were used as a reference. Growth kinetics were compared via cell proliferation and colony forming-unit (CFU) assays. GPCs and PPCs were characterized via cytometric expression of stromal markers, multi-lineage (osteogenic, adipogenic and chondrogenic) differentiation potential, and secretory cytokine profiles. 3-D sphere cultures of GPCs and PPCs were established, and the expression of stemness- and osteogenesis-related markers in 3-D and monolayer cultures was evaluated via gene expression and immunocytochemistry. Results: GPCs and PPCs in both FBS and PL showed characteristic fibroblastic morphology and stromal phenotype (highly positive for CD105/CD90/CD73 and negative for CD34/CD45/HLA-DR). Cell proliferation and CFU efficiency were superior in PL compared to FBS. GPCs and PPCs expanded in both PL and FBS showed multi-lineage differentiation comparable to BMSCs; notably, osteogenic differentiation was enhanced in GPCs expanded in PL. 3-D spheres of GPCs and PPCs were formed and maintained for up to 7 days in xeno-free suspension culture. Notably, expression of stemness- (Sox2, Oct4, Nanog) and osteogenesis-related markers (Runx2, Osx, BMP2) was significantly upregulated in GPC- and PPC-derived spheres vs. monolayers. Conclusions: PPCs and particularly GPCs, due to easy access, cultured in 3-D xeno-free conditions represents a promising strategy for P-/BTE. Student Presenter This abstract is based on research that was funded entirely or partially by an outside source: International Team for Implantology (117/2015), Helse Vest Norway (912048) Disclosure Statement: The submitter must disclose the names of the organizations with which any author have a relationship, the nature of the relationship, and the clinical or research area involved. The following is submitted: NONE
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| 6. |
- Shanbhag, Siddharth, et al.
(författare)
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Efficacy of Humanized Mesenchymal Stem Cell Cultures for Bone Tissue Engineering : A Systematic Review with a Focus on Platelet Derivatives
- 2017
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Ingår i: Tissue engineering. Part B, Reviews. - : Mary Ann Liebert. - 1937-3368 .- 1937-3376. ; 23:6, s. 552-569
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Tidskriftsartikel (refereegranskat)abstract
- Fetal bovine serum (FBS) is the most commonly used supplement for ex vivo expansion of human mesenchymal stem cells (hMSCs) for bone tissue engineering applications. However, from a clinical standpoint, it is important to substitute animal-derived products according to current good manufacturing practice (cGMP) guidelines. Humanized alternatives to FBS include three categories of products: human serum (HS), human platelet derivatives (HPDs)-including platelet lysate (PL) or platelet releasate (PR), produced by freeze/thawing or chemical activation of platelet concentrates, respectively, and chemically defined media (serum-free) (CDM). In this systematic literature review, the in vitro and in vivo osteogenic potential of hMSCs expanded in humanized (HS-, HPD-, or CDM-supplemented) media versus hMSCs expanded in FBS-supplemented media, was compared. In addition, PL and PR were compared in terms of their growth factor (GF)/cytokine-content and cell-culture efficacy. When using either 10-20% autologous or pooled HS, 3-10% pooled HPDs or CDM supplemented with GFs, in comparison with 10-20% FBS, a majority of studies reported similar or superior in vitro proliferation and osteogenic differentiation, and in vivo bone formation in ectopic or orthotopic rodent models. Moreover, a trend for higher GF content was observed in PL versus PR, although evidence for cell culture efficacy is limited. In summary, humanized supplements seem at least equally effective as FBS for hMSC expansion and osteogenic differentiation. Although pooled HPDs appear to be the most favorable supplement for large-scale hMSC expansion, further efforts are needed to standardize the preparation and composition of these products in compliance with cGMP standards.
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| 7. |
- Shanbhag, Siddharth, et al.
(författare)
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Influence of Bone Substitutes on Mesenchymal Stromal Cells in an Inflammatory Microenvironment
- 2023
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Ingår i: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 24:1
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Tidskriftsartikel (refereegranskat)abstract
- Bone regeneration is driven by mesenchymal stromal cells (MSCs) via their interactions with immune cells, such as macrophages (MPs). Bone substitutes, e.g., bi-calcium phosphates (BCPs), are commonly used to treat bone defects. However, little research has focused on MSC responses to BCPs in the context of inflammation. The objective of this study was to investigate whether BCPs influence MSC responses and MSC-MP interactions, at the gene and protein levels, in an inflammatory microenvironment. In setup A, human bone marrow MSCs combined with two different BCP granules (BCP 60/40 or BCP 20/80) were cultured with or without cytokine stimulation (IL1 beta + TNF alpha) to mimic acute inflammation. In setup B, U937 cell-line-derived MPs were introduced via transwell cocultures to setup A. Monolayer MSCs with and without cytokine stimulation served as controls. After 72 h, the expressions of genes related to osteogenesis, healing, inflammation and remodeling were assessed in the MSCs via quantitative polymerase chain reactions. Additionally, MSC-secreted cytokines related to healing, inflammation and chemotaxis were assessed via multiplex immunoassays. Overall, the results indicate that, under both inflammatory and non-inflammatory conditions, the BCP granules significantly regulated the MSC gene expressions towards a pro-healing genotype but had relatively little effect on the MSC secretory profiles. In the presence of the MPs (coculture), the BCPs positively regulated both the gene expression and cytokine secretion of the MSCs. Overall, similar trends in MSC responses were observed with BCP 60/40 and BCP 20/80. In summary, within the limits of in vitro models, these findings suggest that the presence of BCP granules at a surgical site may not necessarily have a detrimental effect on MSC-mediated wound healing, even in the event of inflammation.
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| 8. |
- Shanbhag, Siddharth, et al.
(författare)
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Spheroid Coculture of Human Gingiva-Derived Progenitor Cells With Endothelial Cells in Modified Platelet Lysate Hydrogels
- 2021
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Ingår i: Frontiers in Bioengineering and Biotechnology. - : Frontiers Media S.A.. - 2296-4185. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- Cell coculture strategies can promote angiogenesis within tissue engineering constructs. This study aimed to test the angiogenic potential of human umbilical vein endothelial cells (HUVEC) cocultured with gingiva-derived progenitor cells (GPC) as spheroids in a xeno-free environment. Human platelet lysate (HPL) was used as a cell culture supplement and as a hydrogel matrix (HPLG) for spheroid encapsulation. HUVEC and HUVEC + GPC (1:1 or 5:1) spheroids were encapsulated in various HPLG formulations. Angiogenesis was assessed via in vitro sprouting and in vivo chick chorioallantoic membrane (CAM) assays. HUVEC revealed characteristic in vitro sprouting in HPL/HPLG and this was significantly enhanced in cocultures with GPC (p < 0.05). A trend for greater sprouting was observed in 5:1 vs 1:1 HUVEC + GPC spheroids and in certain HPLG formulations (p > 0.05). Both HUVEC and HUVEC + GPC spheroids in HPLG revealed abundant and comparable neoangiogenesis in the CAM assay (p > 0.05). Spheroid coculture of HUVEC + GPC in HPLG represents a promising strategy to promote angiogenesis.
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| 9. |
- Shanbhag, Siddharth, et al.
(författare)
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Xeno-Free Spheroids of Human Gingiva-Derived Progenitor Cells for Bone Tissue Engineering
- 2020
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Ingår i: Frontiers in Bioengineering and Biotechnology. - : Frontiers Media S.A.. - 2296-4185. ; 8
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Tidskriftsartikel (refereegranskat)abstract
- Gingiva has been identified as a minimally invasive source of multipotent progenitor cells (GPCs) for use in bone tissue engineering (BTE). To facilitate clinical translation, it is important to characterize GPCs in xeno-free cultures. Recent evidence indicates several advantages of three-dimensional (3D) spheroid cultures of mesenchymal stromal cells (MSCs) over conventional 2D monolayers. The present study aimed to characterize human GPCs in xeno-free 2D cultures, and to test their osteogenic potential in 3D cultures, in comparison to bone marrow MSCs (BMSCs). Primary GPCs and BMSCs were expanded in human platelet lysate (HPL) or fetal bovine serum (FBS) and characterized based onin vitroproliferation, immunophenotype and multi-lineage differentiation. Next, 3D spheroids of GPCs and BMSCs were formed via self-assembly and cultured in HPL. Expression of stemness- (SOX2, OCT4, NANOG) and osteogenesis-related markers (BMP2, RUNX2, OPN, OCN) was assessed at gene and protein levels in 3D and 2D cultures. The cytokine profile of 3D and 2D GPCs and BMSCs was assessed via a multiplex immunoassay. Monolayer GPCs in both HPL and FBS demonstrated a characteristic MSC-like immunophenotype and multi-lineage differentiation; osteogenic differentiation of GPCs was enhanced in HPL vs. FBS. CD271(+)GPCs in HPL spontaneously acquired a neuronal phenotype and strongly expressed neuronal/glial markers. 3D spheroids of GPCs and BMSCs with high cell viability were formed in HPL media. Expression of stemness- and osteogenesis-related genes was significantly upregulated in 3D vs. 2D GPCs/BMSCs; the latter was independent of osteogenic induction. Synthesis of SOX2, BMP2 and OCN was confirmed via immunostaining, andin vitromineralization via Alizarin red staining. Finally, secretion of several growth factors and chemokines was enhanced in GPC/BMSC spheroids, while that of pro-inflammatory cytokines was reduced, compared to monolayers. In summary, monolayer GPCs expanded in HPL demonstrate enhanced osteogenic differentiation potential, comparable to that of BMSCs. Xeno-free spheroid culture further enhances stemness- and osteogenesis-related gene expression, and cytokine secretion in GPCs, comparable to that of BMSCs.
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| 10. |
- Suliman, Salwa, et al.
(författare)
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Establishment of a bioluminescence model for microenvironmentally induced oral carcinogenesis with implications for screening bioengineered scaffolds
- 2016
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Ingår i: Head and Neck. - : Wiley-Blackwell. - 1043-3074 .- 1097-0347. ; 38, s. E1177-E1187
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Tidskriftsartikel (refereegranskat)abstract
- Background. Microenvironmental cues play a major role in head and neck cancer. Biodegradable scaffolds used for bone regeneration might also act as stimulative cues for head and neck cancer. The purpose of this study was to establish an experimental model for precise and noninvasive evaluation of tumorigenic potential of microenvironmental cues in head and neck cancer. Methods. Bioluminescence was chosen to image tumor formation. Early neoplastic oral keratinocyte (DOK) cells were luciferase-transduced (DOKLuc), then tested in nonobese diabetic severe combined immunodeficient IL2r gamma(null) mice either orthotopically (tongue) or subcutaneously for their potential as "screening sensors" for diverse microenvironmental cues. Results. Tumors formed after inoculation of DOKLuc were monitored easier by bioluminescence, and bioluminescence was more sensitive in detecting differences between various microenvironmental cues when compared to manual measurements. Development of tumors from DOKLuc grown on scaffolds was also successfully monitored noninvasively by bioluminescence. Conclusion. The model presented here is a noninvasive and sensitive model for monitoring the impact of various microenvironmental cues on head and neck cancer in vivo.
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| 11. |
- Suliman, Salwa, et al.
(författare)
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Immune-instructive copolymer scaffolds using plant-derived nanoparticles to promote bone regeneration
- 2022
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Ingår i: Inflammation and Regeneration. - : Springer Nature. - 1880-8190. ; 42:1
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Tidskriftsartikel (refereegranskat)abstract
- Background Age-driven immune signals cause a state of chronic low-grade inflammation and in consequence affect bone healing and cause challenges for clinicians when repairing critical-sized bone defects in elderly patients. Methods Poly(l-lactide-co-e-caprolactone) (PLCA) scaffolds are functionalized with plant-derived nanoparticles from potato, rhamnogalacturonan-I (RG-I), to investigate their ability to modulate inflammation in vitro in neutrophils and macrophages at gene and protein levels. The scaffolds' early and late host response at gene, protein and histological levels is tested in vivo in a subcutaneous rat model and their potential to promote bone regeneration in an aged rodent was tested in a critical-sized calvaria bone defect. Significant differences were tested using one-way ANOVA, followed by a multiple-comparison Tukey's test with a p value <= 0.05 considered significant. Results Gene expressions revealed PLCA scaffold functionalized with plant-derived RG-I with a relatively higher amount of galactose than arabinose (potato dearabinated (PA)) to reduce the inflammatory state stimulated by bacterial LPS in neutrophils and macrophages in vitro. LPS-stimulated neutrophils show a significantly decreased intracellular accumulation of galectin-3 in the presence of PA functionalization compared to Control (unmodified PLCA scaffolds). The in vivo gene and protein expressions revealed comparable results to in vitro. The host response is modulated towards anti-inflammatory/ healing at early and late time points at gene and protein levels. A reduced foreign body reaction and fibrous capsule formation is observed when PLCA scaffolds functionalized with PA were implanted in vivo subcutaneously. PLCA scaffolds functionalized with PA modulated the cytokine and chemokine expressions in vivo during early and late inflammatory phases. PLCA scaffolds functionalized with PA implanted in calvaria defects of aged rats downregulating pro-inflammatory gene markers while promoting osteogenic markers after 2 weeks in vivo. Conclusion We have shown that PLCA scaffolds functionalized with plant-derived RG-I with a relatively higher amount of galactose play a role in the modulation of inflammatory responses both in vitro and in vivo subcutaneously and promote the initiation of bone formation in a critical-sized bone defect of an aged rodent. Our study addresses the increasing demand in bone tissue engineering for immunomodulatory 3D scaffolds that promote osteogenesis and modulate immune responses.
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| 12. |
- Suliman, Salwa, et al.
(författare)
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In Vivo Host Response and Degradation of Copolymer Scaffolds Functionalized with Nanodiamonds and Bone Morphogenetic Protein 2
- 2016
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Ingår i: Advanced Healthcare Materials. - : Wiley-Blackwell. - 2192-2640 .- 2192-2659. ; 5:6, s. 730-742
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Tidskriftsartikel (refereegranskat)abstract
- The aim is to evaluate the effect of modifying poly[(L-lactide)-co-(epsilon-caprolactone)] scaffolds (PLCL) with nanodiamonds (nDP) or with nDP+physisorbed BMP-2 (nDP+BMP-2) on in vivo host tissue response and degradation. The scaffolds are implanted subcutaneously in Balb/c mice and retrieved after 1, 8, and 27 weeks. Molecular weight analysis shows that modified scaffolds degrade faster than the unmodified. Gene analysis at week 1 shows highest expression of proinflammatory markers around nDP scaffolds; although the presence of inflammatory cells and foreign body giant cells is more prominent around the PLCL. Tissue regeneration markers are highly expressed in the nDP+BMP-2 scaffolds at week 8. A fibrous capsule is detectable by week 8, thinnest around nDP scaffolds and at week 27 thickest around PLCL scaffolds. mRNA levels of ALP, COL1 alpha 2, and ANGPT1 are signifi cantly upregulating in the nDP+BMP-2 scaffolds at week 1 with ectopic bone seen at week 8. Even when almost 90% of the scaffold is degraded at week 27, nDP are observable at implantation areas without adverse effects. In conclusion, modifying PLCL scaffolds with nDP does not aggravate the host response and physisorbed BMP-2 delivery attenuates infl ammation while lowering the dose of BMP-2 to a relatively safe and economical level.
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| 13. |
- Suliman, Salwa, et al.
(författare)
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Release and bioactivity of bone morphogenetic protein-2 are affected by scaffold binding techniques in vitro and in vivo
- 2015
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Ingår i: Journal of Controlled Release. - : Elsevier BV. - 0168-3659 .- 1873-4995. ; 197, s. 148-157
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Tidskriftsartikel (refereegranskat)abstract
- A low dose of 1 mu g rhBMP-2 was immobilised by four different functionalising techniques on recently developed poly(L-lactide)-co-(epsilon-caprolactone) [(poly(LLA-co-CL)] scaffolds. It was either (i) physisorbed on unmodified scaffolds [PHY], (ii) physisorbed onto scaffolds modified with nanodiamond particles [nDP-PHY], (iii) covalently linked onto nDPs that were used to modify the scaffolds [nDP-COV] or (iv) encapsulated in microspheres distributed on the scaffolds [MICS]. Release kinetics of BMP-2 from the different scaffolds was quantified using targeted mass spectrometry for up to 70 days. PHY scaffolds had an initial burst of release while MICS showed a gradual and sustained increase in release. In contrast, NDP-PHY and nDP-COV scaffolds showed no significant release, although nDP-PHY scaffolds maintained bioactivity of BMP-2. Human mesenchymal stem cells cultured in vitro showed upregulated BMP-2 and osteocalcin gene expression at both week 1 and week 3 in the MICS and nDP-PHY scaffold groups. These groups also demonstrated the highest BMP-2 extracellular protein levels as assessed by ELISA, and mineralization confirmed by Alizarin red. Cells grown on the PHY scaffolds in vitro expressed collagen type 1 alpha 2 early but the scaffold could not sustain rhBMP-2 release to express mineralization. After 4 weeks post-implantation using a rat mandible critical-sized defect model, micro-CT and Masson trichrome results showed accelerated bone regeneration in the PHY, nDP-PHY and MICS groups. The results demonstrate that PHY scaffolds may not be desirable for clinical use, since similar osteogenic potential was not seen under both in vitro and in vivo conditions, in contrast to nDP-PHY and MICS groups, where continuous low doses of BMP-2 induced satisfactory bone regeneration in both conditions. The nDP-PHY scaffolds used here in critical-sized bone defects for the first time appear to have promise compared to growth factors adsorbed onto a polymer alone and the short distance effect prevents adverse systemic side effects.
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