| 1. |
- Alakpa, Enateri V., et al.
(författare)
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Bioprinted schwann and mesenchymal stem cell co-cultures for enhanced spatial control of neurite outgrowth
- 2023
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Ingår i: Gels. - : MDPI. - 2310-2861. ; 9:3
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Tidskriftsartikel (refereegranskat)abstract
- Bioprinting nerve conduits supplemented with glial or stem cells is a promising approach to promote axonal regeneration in the injured nervous system. In this study, we examined the effects of different compositions of bioprinted fibrin hydrogels supplemented with Schwann cells and mesenchymal stem cells (MSCs) on cell viability, production of neurotrophic factors, and neurite outgrowth from adult sensory neurons. To reduce cell damage during bioprinting, we analyzed and optimized the shear stress magnitude and exposure time. The results demonstrated that fibrin hydrogel made from 9 mg/mL of fibrinogen and 50IE/mL of thrombin maintained the gel’s highest stability and cell viability. Gene transcription levels for neurotrophic factors were significantly higher in cultures containing Schwann cells. However, the amount of the secreted neurotrophic factors was similar in all co-cultures with the different ratios of Schwann cells and MSCs. By testing various co-culture combinations, we found that the number of Schwann cells can feasibly be reduced by half and still stimulate guided neurite outgrowth in a 3D-printed fibrin matrix. This study demonstrates that bioprinting can be used to develop nerve conduits with optimized cell compositions to guide axonal regeneration.
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| 2. |
- Anerillas, Luis Oliveros, et al.
(författare)
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Platelet lysate for expansion or osteogenic differentiation of bone marrow mesenchymal stem cells for 3D tissue constructs
- 2023
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Ingår i: Regenerative Therapy. - : Japanese Society of Regenerative Medicine. - 2352-3204. ; 24, s. 298-310
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Tidskriftsartikel (refereegranskat)abstract
- Background: The use of mesenchymal stem cells (MSCs) for the development of tissue-engineered constructs has advanced in recent years. However, future clinically approved products require following good manufacturing practice (GMP) guidelines. This includes using alternatives to xenogeneic-derived cell culture supplements to avoid rejection of the transplants. Consequently, human platelet lysate (PLT) has been adopted as an affordable and effective alternative to foetal bovine serum (FBS) in traditional 2D cultures. However, little is known about its effect in more advanced 3D culture systems.Methods: We evaluated bone marrow MSCs (BMSCs) proliferation and CD marker expression in cells expanded in FBS or PLT-supplemented media. Differentiation capacity of the BMSCs expanded in the presence of the different supplements was evaluated in 3D type I collagen hydrogels. Furthermore, the effects of the supplements on the process of differentiation were analyzed by using qPCR and histological staining.Results: Cell proliferation was greater in PLT-supplemented media versus FBS. BMSCs expanded in PLT showed similar osteogenic differentiation capacity in 3D compared with FBS expanded cells. In contrast, when cells were 3D differentiated in PLT they showed lower osteogenesis versus the traditional FBS protocol. This was also the case for adipogenic differentiation, in which FBS supplementation was superior to PLT.Conclusions: PLT is a superior alternative to FBS for the expansion of MSCs without compromising their subsequent differentiation capacity in 3D. However, differentiation in PLT is impaired. Thus, PLT can be used to reduce the time required to expand the necessary cell numbers for development of 3D tissue engineered MSC constructs.
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| 3. |
- Anerillas, Luis Oliveros, et al.
(författare)
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Three-dimensional osteogenic differentiation of bone marrow mesenchymal stem cells promotes matrix metallopeptidase 13 (Mmp13) expression in type i collagen hydrogels
- 2021
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Ingår i: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 22:24
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Tidskriftsartikel (refereegranskat)abstract
- Autologous bone transplantation is the principal method for reconstruction of large bone defects. This technique has limitations, such as donor site availability, amount of bone needed and morbidity. An alternative to this technique is tissue engineering with bone marrow-derived mesenchymal stem cells (BMSCs). In this study, our aim was to elucidate the benefits of culturing BMSCs in 3D compared with the traditional 2D culture. In an initial screening, we combined BMSCs with four different biogels: unmodified type I collagen (Col I), type I collagen methacrylate (ColMa), an alginate and cellulose-based bioink (CELLINK) and a gelatin-based bioink containing xanthan gum (GelXA-bone). Col I was the best for structural integrity and maintenance of cell morphology. Osteogenic, adipogenic, and chondrogenic differentiations of the BMSCs in 2D versus 3D type I collagen gels were investigated. While the traditional pellet culture for chondrogenesis was superior to our tested 3D culture, Col I hydrogels (i.e., 3D) favored adipogenic and osteogenic differentiation. Further focus of this study on osteogenesis were conducted by comparing 2D and 3D differentiated BMSCs with Osteoimage® (stains hydroxyapatite), von Kossa (stains anionic portion of phosphates, carbonates, and other salts) and Alizarin Red (stains Ca2+ deposits). Multivariate gene analysis with various covariates showed low variability among donors, successful osteogenic differentiation, and the identification of one gene (matrix metallopeptidase 13, MMP13) significantly differentially expressed in 2D vs. 3D cultures. MMP13 protein expression was confirmed with immunohistochemistry. In conclusion, this study shows evidence for the suitability of type I collagen gels for 3D osteogenic differentiation of BMSCs, which might improve the production of tissue-engineered constructs for treatment of bone defects.
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| 4. |
- Kelk, Peyman, et al.
(författare)
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Aggregatibacter actinomycetemcomitans Leukotoxin Activates the NLRP3 Inflammasome and Cell-to-Cell Communication
- 2022
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Ingår i: Pathogens. - Basel : MDPI. - 2076-0817. ; 11:2
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Tidskriftsartikel (refereegranskat)abstract
- Carriers of highly leukotoxic genotypes of Aggregatibacter actinomycetemcomitans are at high risk for rapid degradation of tooth-supporting tissues. The leukotoxin (LtxA) expressed by this bacterium induces a rapid pro-inflammatory response in leukocytes that results in cell death. The aim of the present study was to increase the understanding of LtxA-induced leukocyte activation mechanisms and of possible associated osteoclast differentiation. The effect of LtxA on activation of the inflammasome complex was studied in THP-1 wild type and in NLRP3- and ASC knockout cells. Cell-to-cell communication was assessed by fluorescent parachute assays, and THP-1 differentiation into osteoclast-like cells was investigated microscopically. The results showed that LtxA induced inflammatory cell death, which involved activation of the NLRP3 inflammasome and gap junction cell-to-cell communication. THP-1 cells treated with lipopolysaccharide (LPS) and LtxA together differentiated into an osteoclast-like phenotype. Here, LPS prevented LtxA-mediated cell death but failed to induce osteoclast differentiation on its own. However, pit formation was not significantly enhanced by LtxA. We conclude that A. actinomycetemcomitans leukotoxicity mediates activation of the NLRP3 inflammasome and cell-to-cell communication in the induced pro-inflammatory cell death. In addition, LtxA stimulated differentiation towards osteoclasts-like cells in LPS-treated THP-1 cells
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| 5. |
- Kelk, Peyman, et al.
(författare)
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Successful complete oral rehabilitation of a patient with osteopetrosis with extensive pre-treatments, bone grafts, dental implants and fixed bridges: a multidisciplinary case report.
- 2023
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Ingår i: BMC oral health. - : BioMed Central (BMC). - 1472-6831. ; 23:1
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Tidskriftsartikel (refereegranskat)abstract
- Osteopetrosis comprises a group of inherited disorders that are rare and result in abnormal bone structure. Bone remodeling is extremely inhibited because osteoclasts are nonfunctional or lacking. This condition causes overgrowth of bone with disappearance of the bone marrow, leading to aplastic anemia; obstruction of nerve passages in the skull leads to blindness and often hearing impairment. In most cases, osteopetrosis results in oral complications such as tooth deformation, hypomineralization, and delayed or absent tooth eruption. The only curative treatment is hematopoietic stem cell transplantation (HSCT). The main treatment of the oral complications during childhood and adolescence consists in protecting the erupted teeth against caries disease through prophylactic treatment aimed at optimal oral hygiene through frequent regular dental visits throughout life. Many patients with osteopetrosis require major oral rehabilitation to treat complications of the disease. Improved results of HSCT increase the likelihood that dental professionals will encounter patients with osteopetrosis.In this case report, we show that individuals with osteopetrosis who have severe oral complications can be treated successfully if they are treated for osteopetrosis at an early age. The boy had his dental care in pedodontics, and regular multidisciplinary meetings were held for future treatment planning. At the age of 15, he was then referred for rehabilitation. The initial evaluations revealed no further growth in the alveolar bone. The rehabilitation was done stepwise, with extraction of malformed and malpositioned teeth. Initially, the patient received a removable partial denture followed by reconstruction of the width of the alveolar process, titanium implants, temporary fixed bridges, and finally screw-retained titanium-ceramic bridges with titanium frames for the upper and lower jaws.The three-year follow-up after loading indicated a stable marginal bone level and optimal oral hygiene as a result of frequent professional oral hygiene care. The patient showed no signs of symptoms from the temporomandibular joint and has adapted to the new jaw relation without any functional or phonetical issues.
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| 6. |
- Kumar Kuna, Vijay, et al.
(författare)
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Efficacy of Nerve-Derived Hydrogels to Promote Axon Regeneration Is Influenced by the Method of Tissue Decellularization
- 2022
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Ingår i: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 23:15
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Tidskriftsartikel (refereegranskat)abstract
- Injuries to large peripheral nerves are often associated with tissue defects and require reconstruction using autologous nerve grafts, which have limited availability and result in donor site morbidity. Peripheral nerve-derived hydrogels could potentially supplement or even replace these grafts. In this study, three decellularization protocols based on the ionic detergents sodium dodecyl sulfate (P1) and sodium deoxycholate (P2), or the organic solvent tri-n-butyl phosphate (P3), were used to prepare hydrogels. All protocols resulted in significantly decreased amounts of genomic DNA, but the P2 hydrogel showed the best preservation of extracellular matrix proteins, cytokines, and chemokines, and reduced levels of sulfated glycosaminoglycans. In vitro P1 and P2 hydrogels supported Schwann cell viability, secretion of VEGF, and neurite outgrowth. Surgical repair of a 10 mm-long rat sciatic nerve gap was performed by implantation of tubular polycaprolactone conduits filled with hydrogels followed by analyses using diffusion tensor imaging and immunostaining for neuronal and glial markers. The results demonstrated that the P2 hydrogel considerably increased the number of axons and the distance of regeneration into the distal nerve stump. In summary, the method used to decellularize nerve tissue affects the efficacy of the resulting hydrogels to support regeneration after nerve injury.
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| 7. |
- Lauvrud, Anne Therese, 1975-
(författare)
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Optimizing stem cells for reconstructive surgery
- 2024
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Fat grafting has become an established method in plastic surgery for treating soft tissue defects. The results for survival of the fat being transplanted is unpredictable and supplementation of the graft with the Stromal Vascular Fraction (SVF) or cultures Adipose tissue-derived stem cells (ASCs) can enhance graft viability. The ASCs are a heterogenous group of cells with various cell membrane markers, and differing growth promoting and differentiation characteristics of the stem cells derived from the fat. It is of high importance when expanding cells prior to the transplantation of the cells into patients, that the culture conditions are well defined and ideally are xenofree, avoiding use of animal-derived products. Furthermore, the procedures must be safe and not increase risk for recurrence of cancer after reconstructive surgeries. This thesis explores the phenotypic properties of a selected population of ASCs, with a view to determining their suitability for transplantation into fat grafts. ASCs were isolated from SVF of human abdominal fat and CD146+ cells were selected using immunomagnetic beads. The proliferation, angiogenic and adipogenic properties were significantly higher in the CD146+ cells. Stem cells were also isolated from lipoaspirate obtained using two different liposuction methods. Waterjet lipoaspirates yielded the greatest number of CD146+ cells with high adipogenic potential and angiogenic activity. The cells could also be successfully isolated using a closed processing system. Cells were expanded in either foetal bovine serum, platelet lysate or a chemically defined xenofree (XV) medium. Cultures in XV medium proliferated the fastest, expressed the highest number of CD146+ cells, and showed the best adipogenic and angiogenic properties. To test possible ASCs interactions with cancer cells, co-cultures with MCF-7 breast cancer cells were established. Conditioned medium from co-cultures significantly increased the migration of the cancer cells but not their proliferation, and there was increased expression of Tenascin-C in these cultures. The research in this thesis work has shown more optimal ways to isolate and expand ASCs, potentially offering new therapeutic reconstructive treatment options for a variety of medical conditions.
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| 8. |
- Lauvrud, Anne Therese, et al.
(författare)
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Water jet-assisted lipoaspiration and Sepax cell separation system for the isolation of adipose stem cells with high adipogenic potential
- 2021
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Ingår i: Journal of Plastic, Reconstructive & Aesthetic Surgery. - : Elsevier. - 1748-6815 .- 1878-0539. ; 74:10, s. 2759-2767
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Tidskriftsartikel (refereegranskat)abstract
- Introduction: Water jet-assisted liposuction has gained popularity due to favourable fat grafting outcomes. In this study, we compared stem cells obtained from fat isolated with manual or the water jet-assisted procedure.Methods: Liposuction of abdominal fat was performed using the two methods on each donor (n = 10). Aspirate samples were collagenase digested and the isolated cells seeded in vitro prior to proliferation, adipogenic differentiation and angiogenic activity analyses.Results: Cells from either procedure proliferated at similar rates and exhibited a similar colony-forming ability. The cells expressed stem cell markers CD73, CD90 and CD105. In the water jet cell preparations, there were higher numbers of cells expressing CD146. Robust adipogenic differentiation was observed in cultures expanded from both manual and water jet lipoaspirates. Gene analysis showed higher expression of the adipocyte markers aP2 and GLUT4 in the adipocyte-differentiated water jet cell preparations, and ELISA indicated increased secretion of adiponectin from these cells. Both cell groups expressed vasculogenic factors and the water jet cells promoted the highest levels of in vitro angiogenesis. Given these positive results, we further characterised the water jet cells when prepared using an automated closed cell processing unit, the Sepax-2 system (Cytiva). The growth and stem cell properties of the Sepax-processed cells were similar to the standard centrifugation protocol, but there was evidence for greater adipogenic differentiation in the Sepax-processed cells.Conclusions: Water jet lipoaspirates yield cells with high adipogenic potential and angiogenic activity, which may be beneficial for use in cell-assisted lipotransfers.
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| 9. |
- Oliveros Anerillas, Luis, 1995-
(författare)
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Development and optimization of a 3D in vitro model for osteogenic biomaterial evaluation
- 2023
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Despite the innate regenerative capabilities of bone tissue, self-repair is impaired when an injury exceeds the critical size threshold because of trauma, congenital, or pathological conditions. Autologous transplantation is the gold standard to reconstruct large bone defects. However, this method has drawbacks such as limited amount of graftable material, limited accessibility, and donor site morbidity. For these reasons, alternative regenerative medicine and tissue engineering approaches are being developed, including implantable scaffolds.The use of in vitro-made scaffolds containing biomaterials that mimic the functional characteristics and composition of extracellular bone matrix has been favored in 3D vs 2D in vitro culture systems. Adult stem cells such as mesenchymal stem cells (MSCs), that give rise to bone building cells, have been used in combination with various biomaterials. The development of an implantable scaffold with or without cells requires extensive in vitro validation and optimization prior to its testing in vivo. Thus, the primary aim of this thesis was to develop a 3D model for the optimization of MSC differentiation. Further aims were to utilize this 3D model to evaluate the MSC response to a novel osteogenic biomaterial. To achieve these objectives human bone marrow MSCs (BMSCs) were utilized in various hydrogels in combination with chemical differentiation factors or biomaterials. Moreover, the osteogenic capability of the tested biomaterials and their induced inflammatory/angiogenic responses were investigated, and the culture conditions were optimized for clinical application. In this thesis, a comparison between 2D and 3D (hydrogels) in vitro culture models was developed with the purpose of studying osteogenic differentiation in BSMCs. Testing various hydrogels revealedt he superiority of type I collagen hydrogels for the osteogenic 3D in vitro culture system. Further, cell culture conditions were improved for the expansion and differentiation of BMSCs to fulfill clinically approved standards according to Good Manufacturing Practice (GMP) conditions. Comparisons between fetal bovine serum (FBS) and human platelet lysate (PLT) showed superior cellular differentiation in FBS, while PLT enhanced cell proliferation. Based on the developed 3D model, the osteogenic properties of a novel nanoporous silica calcium phosphate (nSCP) material were investigated. The results indicated that nSCP was osteoinductive, involving different pathways compared with the traditional chemical differentiation protocols or other tested osteogenic biomaterials. Finally, the inflammatory and angiogenic responses from human BMSCs and an immortalized monocyte cell line (THP-1) exposed to nSCP in the established 3D model were assessed.The results indicated limited inflammatory effect of nSCP, while inducing the secretion of pro-angiogenic cytokines. The bioactivity of these released factors was confirmed in an assay using human endothelial cells.Taken together, this thesis presents a 3D in vitro model for studying osteogenic differentiation in MSCs, which can be utilized to evaluate, validate, and optimize biomaterial candidates for bone regeneration applications.
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| 10. |
- Qu, Chengjuan, 1967-, et al.
(författare)
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Evaluation of growth, stemness, and angiogenic properties of dental pulp stem cells cultured in cGMP xeno-/serum-free medium
- 2020
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Ingår i: Cell and Tissue Research. - : Springer. - 0302-766X .- 1432-0878. ; 380, s. 93-105
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Tidskriftsartikel (refereegranskat)abstract
- This study was aimed to investigate the effects of cGMP xeno-/serum-free medium (XSF, Irvine Scientific) on the properties of human dental pulp stem cells (DPSCs). DPSCs, from passage 2, were cultured in XSF or fetal bovine serum (FBS)-supplemented medium, and sub-cultured up to passage 8. Cumulative population doublings (PDs) and the number of colony-forming-units (CFUs) were determined. qRT-PCR, ELISA, and in vitro assays were used to assess angiogenic capacity. Flow cytometry was used to measure CD73, CD90, and CD105 expression. Differentiation into osteo-, adipo-, and chondrogenic cell lineages was performed. DPSCs showed more elongated morphology, a reduced rate of proliferation at later passages, and lower CFU counts in XSF compared with FBS. Expression of angiogenic factors at the gene and protein levels varied in the two media and with passage number, but cells grown in XSF had more in vitro angiogenic activity. The majority of early and late passage DPSCs cultured in XSF expressed CD73 and CD90. In contrast, the percentage of CD105 positive DPSCs in XSF medium was significantly lower with increased passage whereas the majority of cells cultured in FBS were CD105 positive. Switching XSF-cultured DPSCs to medium supplemented with human serum restored the expression of CD105. The tri-lineage differentiation of DPSCs cultured under XSF and FBS conditions was similar. We showed that despite reduced CD105 expression levels, DPSCs expanded in XSF medium maintained a functional MSC phenotype. Furthermore, restoration of CD105 expression is likely to occur upon in vivo transplantation, when cells are exposed to human serum.
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