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13 pages, 3597 KiB

Open AccessArticle

The Number of Platelets in Patient’s Blood Influences the Mechanical and Morphological Properties of PRP-Clot and Lysophosphatidic Acid Quantity in PRP

by Michela Bosetti, Paolo Boffano, Alice Marchetti, Massimiliano Leigheb, Mattia Colli and Matteo Brucoli

Int. J. Mol. Sci. 2020, 21(1), 139; https://doi.org/10.3390/ijms21010139 - 24 Dec 2019

Cited by 7 | Viewed by 2783

Abstract

The objectives of this study were to compare platelet-rich plasma (PRP) from patients with different concentrations of platelets and to assess the influence of these PRP preparations on human osteoblast (hOB) activity. In the literature, growth factors released by activated platelets have been [...] Read more.

The objectives of this study were to compare platelet-rich plasma (PRP) from patients with different concentrations of platelets and to assess the influence of these PRP preparations on human osteoblast (hOB) activity. In the literature, growth factors released by activated platelets have been considered responsible for the active role of PRP on bone regeneration but no specific role has been attributed to lysophosphatidic acid (LPA) as a possible effector of biological responses. In this study, patients were grouped into either group A (poor in platelets) or group B (rich in platelets). Clots from PRP fraction 2 (F2-clots), obtained with CaCl₂ activation of PRP from the two groups, were compared macroscopically and microscopically and for their mechanical properties before testing their activity on the proliferation and migration of hOB. LPA was quantified before and after PRP fractioning and activation. The fibrin network of F2-clots from patients with a lower platelet concentration had an organized structure with large and distinct fibers while F2-clots from patients in group B revealed a similar structure to those in group A but with a slight increase in density. ELISA results showed a significantly higher plasma level of LPA in patients with a higher platelet concentration (group B) in comparison to those in group A (p < 0.05). This different concentration was evidenced in PRP but not in the clots. Depending on the number of platelets in patient’s blood, a PRP-clot with higher or lower mechanical properties can be obtained. The higher level of LPA in PRP from patients richer in platelets should be considered as responsible for the higher hOB activity in bone regeneration. Full article

(This article belongs to the Special Issue Molecular and Tissue Engineering Approaches in Musculoskeletal Regenerative Medicine)

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15 pages, 4517 KiB

Open AccessArticle

Meniscus-Derived Matrix Bioscaffolds: Effects of Concentration and Cross-Linking on Meniscus Cellular Responses and Tissue Repair

by Lucas P. Lyons, Sofia Hidalgo Perea, J. Brice Weinberg, Jocelyn R. Wittstein and Amy L. McNulty

Int. J. Mol. Sci. 2020, 21(1), 44; https://doi.org/10.3390/ijms21010044 - 19 Dec 2019

Cited by 12 | Viewed by 4198

Abstract

Meniscal injuries, particularly in the avascular zone, have a low propensity for healing and are associated with the development of osteoarthritis. Current meniscal repair techniques are limited to specific tear types and have significant risk for failure. In previous work, we demonstrated the [...] Read more.

Meniscal injuries, particularly in the avascular zone, have a low propensity for healing and are associated with the development of osteoarthritis. Current meniscal repair techniques are limited to specific tear types and have significant risk for failure. In previous work, we demonstrated the ability of meniscus-derived matrix (MDM) scaffolds to augment the integration and repair of an in vitro meniscus defect. The objective of this study was to determine the effects of percent composition and dehydrothermal (DHT) or genipin cross-linking of MDM bioscaffolds on primary meniscus cellular responses and integrative meniscus repair. In all scaffolds, the porous microenvironment allowed for exogenous cell infiltration and proliferation, as well as endogenous meniscus cell migration. The genipin cross-linked scaffolds promoted extracellular matrix (ECM) deposition and/or retention. The shear strength of integrative meniscus repair was improved with increasing percentages of MDM and genipin cross-linking. Overall, the 16% genipin cross-linked scaffolds were most effective at enhancing integrative meniscus repair. The ability of the genipin cross-linked scaffolds to attract endogenous meniscus cells, promote glycosaminoglycan and collagen deposition, and enhance integrative meniscus repair reveals that these MDM scaffolds are promising tools to augment meniscus healing. Full article

(This article belongs to the Special Issue Molecular and Tissue Engineering Approaches in Musculoskeletal Regenerative Medicine)

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15 pages, 2013 KiB

Open AccessArticle

Calcium Polyphosphate Nanoparticles Act as an Effective Inorganic Phosphate Source during Osteogenic Differentiation of Human Mesenchymal Stem Cells

by Luan Phelipe Hatt, Keith Thompson, Werner E. G. Müller, Martin James Stoddart and Angela Rita Armiento

Int. J. Mol. Sci. 2019, 20(22), 5801; https://doi.org/10.3390/ijms20225801 - 18 Nov 2019

Cited by 18 | Viewed by 4499

Abstract

The ability of bone-marrow-derived mesenchymal stem/stromal cells (BM-MSCs) to differentiate into osteoblasts makes them the ideal candidate for cell-based therapies targeting bone-diseases. Polyphosphate (polyP) is increasingly being studied as a potential inorganic source of phosphate for extracellular matrix mineralisation. The aim of this [...] Read more.

The ability of bone-marrow-derived mesenchymal stem/stromal cells (BM-MSCs) to differentiate into osteoblasts makes them the ideal candidate for cell-based therapies targeting bone-diseases. Polyphosphate (polyP) is increasingly being studied as a potential inorganic source of phosphate for extracellular matrix mineralisation. The aim of this study is to investigate whether polyP can effectively be used as a phosphate source during the in vitro osteogenic differentiation of human BM-MSCs. Human BM-MSCs are cultivated under osteogenic conditions for 28 days with phosphate provided in the form of organic β-glycerolphosphate (BGP) or calcium-polyP nanoparticles (polyP-NP). Mineralisation is demonstrated using Alizarin red staining, cellular ATP content, and free phosphate levels are measured in both the cells and the medium. The effects of BGP or polyP-NP on alkaline phosphatase (ALP) activity and gene expression of a range of osteogenic-related markers are also assessed. PolyP-NP supplementation displays comparable effects to the classical BGP-containing osteogenic media in terms of mineralisation, ALP activity and expression of osteogenesis-associated genes. This study shows that polyP-NP act as an effective source of phosphate during mineralisation of BM-MSC. These results open new possibilities with BM-MSC-based approaches for bone repair to be achieved through doping of conventional biomaterials with polyP-NP. Full article

(This article belongs to the Special Issue Molecular and Tissue Engineering Approaches in Musculoskeletal Regenerative Medicine)

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18 pages, 2161 KiB

Open AccessArticle

Regulation of Inflammatory Response in Human Osteoarthritic Chondrocytes by Novel Herbal Small Molecules

by Reihane Ziadlou, Andrea Barbero, Martin J. Stoddart, Michael Wirth, Zhen Li, Ivan Martin, Xin-luan Wang, Ling Qin, Mauro Alini and Sibylle Grad

Int. J. Mol. Sci. 2019, 20(22), 5745; https://doi.org/10.3390/ijms20225745 - 15 Nov 2019

Cited by 20 | Viewed by 3527

Abstract

In this study, 34 Traditional Chinese Medicine (TCM) compounds were screened for potential anabolic and anti-inflammatory properties on human osteoarthritic (OA) chondrocytes. The anabolic effects were assessed by measuring the glycosaminoglycan (GAG) relative to the DNA content using a 3D pellet culture model. [...] Read more.

In this study, 34 Traditional Chinese Medicine (TCM) compounds were screened for potential anabolic and anti-inflammatory properties on human osteoarthritic (OA) chondrocytes. The anabolic effects were assessed by measuring the glycosaminoglycan (GAG) relative to the DNA content using a 3D pellet culture model. The most chondrogenic compounds were tested in an inflammatory model consisting of 3 days of treatment with cytokines (IL-1β/TNF-α) with or without supplementation of TCM compounds. The anti-inflammatory effects were assessed transcriptionally, biochemically and histologically. From the 34 compounds, Vanilic acid (VA), Epimedin A (Epi A) and C (Epi C), 2′′-O-rhamnosylicariside II (2-O-rhs II), Icariin, Psoralidin (PS), Protocatechuicaldehyde (PCA), 4-Hydroxybenzoic acid (4-HBA) and 5-Hydroxymethylfurfural (5-HMF) showed the most profound anabolic effects. After induction of inflammation, pro-inflammatory and catabolic genes were upregulated, and GAG/DNA was decreased. VA, Epi C, PS, PCA, 4-HBA and 5-HMF exhibited anti-catabolic and anti-inflammatory effects and prevented the up-regulation of pro-inflammatory markers including metalloproteinases and cyclooxygenase 2. After two weeks of treatment with TCM compounds, the GAG/DNA ratio was restored compared with the negative control group. Immunohistochemistry and Safranin-O staining confirmed superior amounts of cartilaginous matrix in treated pellets. In conclusion, VA, Epi C, PS, PCA, 4-HBA and 5-HMF showed promising anabolic and anti-inflammatory effects. Full article

(This article belongs to the Special Issue Molecular and Tissue Engineering Approaches in Musculoskeletal Regenerative Medicine)

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16 pages, 4688 KiB

Open AccessArticle

Biocompatible Aloe vera and Tetracycline Hydrochloride Loaded Hybrid Nanofibrous Scaffolds for Skin Tissue Engineering

by Hariharan Ezhilarasu, Raghavendra Ramalingam, Chetna Dhand, Rajamani Lakshminarayanan, Asif Sadiq, Chinnasamy Gandhimathi, Seeram Ramakrishna, Boon Huat Bay, Jayarama Reddy Venugopal and Dinesh Kumar Srinivasan

Int. J. Mol. Sci. 2019, 20(20), 5174; https://doi.org/10.3390/ijms20205174 - 18 Oct 2019

Cited by 54 | Viewed by 4348

Abstract

Aloe vera (AV) and tetracycline hydrochloride (TCH) exhibit significant properties such as anti-inflammatory, antioxidant and anti-bacterial activities to facilitate skin tissue engineering. The present study aims to develop poly-ε-caprolactone (PCL)/ AV containing curcumin (CUR), and TCH loaded hybrid nanofibrous scaffolds to validate the [...] Read more.

Aloe vera (AV) and tetracycline hydrochloride (TCH) exhibit significant properties such as anti-inflammatory, antioxidant and anti-bacterial activities to facilitate skin tissue engineering. The present study aims to develop poly-ε-caprolactone (PCL)/ AV containing curcumin (CUR), and TCH loaded hybrid nanofibrous scaffolds to validate the synergistic effect on the fibroblast proliferation and antimicrobial activity against Gram-positive and Gram-negative bacteria for wound healing. PCL/AV, PCL/CUR, PCL/AV/CUR and PCL/AV/TCH hybrid nanofibrous mats were fabricated using an electrospinning technique and were characterized for surface morphology, the successful incorporation of active compounds, hydrophilicity and the mechanical property of nanofibers. SEM revealed that there was a decrease in the fiber diameter (ranging from 360 to 770 nm) upon the addition of AV, CUR and TCH in PCL nanofibers, which were randomly oriented with bead free morphology. FTIR spectra of various electrospun samples confirmed the successful incorporation of AV, CUR and TCH into the PCL nanofibers. The fabricated nanofibrous scaffolds possessed mechanical properties within the range of human skin. The biocompatibility of electrospun nanofibrous scaffolds were evaluated on primary human dermal fibroblasts (hDF) by MTS assay, CMFDA, Sirius red and F-actin stainings. The results showed that the fabricated PCL/AV/CUR and PCL/AV/TCH nanofibrous scaffolds were non-toxic and had the potential for wound healing applications. The disc diffusion assay confirmed that the electrospun nanofibrous scaffolds possessed antibacterial activity and provided an effective wound dressing for skin tissue engineering. Full article

(This article belongs to the Special Issue Molecular and Tissue Engineering Approaches in Musculoskeletal Regenerative Medicine)

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15 pages, 3770 KiB

Open AccessArticle

Osteogenic Differentiation of Mesenchymal Stem Cells with Silica-Coated Gold Nanoparticles for Bone Tissue Engineering

by Chinnasamy Gandhimathi, Ying Jie Quek, Hariharan Ezhilarasu, Seeram Ramakrishna, Boon-Huat Bay and Dinesh Kumar Srinivasan

Int. J. Mol. Sci. 2019, 20(20), 5135; https://doi.org/10.3390/ijms20205135 - 16 Oct 2019

Cited by 28 | Viewed by 5392

Abstract

Multifunctional nanofibrous scaffolds for effective bone tissue engineering (BTE) application must incorporate factors to promote neovascularization and tissue regeneration. In this study, silica-coated gold nanoparticles Au(SiO₂) were tested for their ability to promote differentiation of human mesenchymal stem cells (hMSCs) into [...] Read more.

Multifunctional nanofibrous scaffolds for effective bone tissue engineering (BTE) application must incorporate factors to promote neovascularization and tissue regeneration. In this study, silica-coated gold nanoparticles Au(SiO₂) were tested for their ability to promote differentiation of human mesenchymal stem cells (hMSCs) into osteoblasts. Biocompatible poly-ε-caprolactone (PCL), PCL/silk fibroin (SF) and PCL/SF/Au(SiO₂) loaded nanofibrous scaffolds were first fabricated by an electrospinning method. Electrospun nanofibrous scaffolds were characterized for fiber architecture, porosity, pore size distribution, fiber wettability and the relevant mechanical properties using field emission scanning electron microscopy (FESEM), porosimetry, determination of water contact angle, measurements by a surface analyzer and tabletop tensile-tester measurements. FESEM images of the scaffolds revealed beadless, porous, uniform fibers with diameters in the range of 164 ± 18.65 nm to 215 ± 32.12 nm and porosity of around 88–92% and pore size distribution around 1.45–2.35 µm. Following hMSCs were cultured on the composite scaffolds. Cell-scaffold interaction, morphology and proliferation of were analyzed by FESEM analysis, MTS (3-(4,5-dimethyl thiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt) and CMFDA (5-choromethyl fluorescein acetate) dye assays. Osteogenic differentiation of MSCs into osteogenic cells were determined by alkaline phosphatase (ALP) activity, mineralization by alizarin red S (ARS) staining and osteocalcin expression by immunofluorescence staining. The results revealed that the addition of SF and Au(SiO₂) to PCL scaffolds enhanced the mechanical strength, interconnecting porous structure and surface roughness of the scaffolds. This, in turn, led to successful osteogenic differentiation of hMSCs with improved cell adhesion, proliferation, differentiation, mineralization and expression of pro-osteogenic cellular proteins. This provides huge support for Au(SiO₂) as a suitable material in BTE. Full article

(This article belongs to the Special Issue Molecular and Tissue Engineering Approaches in Musculoskeletal Regenerative Medicine)

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14 pages, 2829 KiB

Open AccessArticle

Impact of Serum Source on Human Mesenchymal Stem Cell Osteogenic Differentiation in Culture

by Alexander Popov, Colin Scotchford, David Grant and Virginie Sottile

Int. J. Mol. Sci. 2019, 20(20), 5051; https://doi.org/10.3390/ijms20205051 - 11 Oct 2019

Cited by 14 | Viewed by 3181

Abstract

Human mesenchymal stem cells (MSCs) show promise for musculoskeletal repair applications. Animal-derived serum is extensively used for MSC culture as a source of nutrients, extracellular matrix proteins and growth factors. However, the routine use of fetal calf serum (FCS) is not innocuous due [...] Read more.

Human mesenchymal stem cells (MSCs) show promise for musculoskeletal repair applications. Animal-derived serum is extensively used for MSC culture as a source of nutrients, extracellular matrix proteins and growth factors. However, the routine use of fetal calf serum (FCS) is not innocuous due to its animal antigens and ill-defined composition, driving the development of alternatives protocols. The present study sought to reduce exposure to FCS via the transient use of human serum. Transient exposure to animal serum had previously proved successful for the osteogenic differentiation of MSCs but had not yet been tested with alternative serum sources. Here, human serum was used to support the proliferation of MSCs, which retained surface marker expression and presented higher alkaline phosphatase activity than those in FCS-based medium. Addition of osteogenic supplements supported strong mineralisation over a 3-week treatment. When limiting serum exposure to the first five days of treatment, MSCs achieved higher differentiation with human serum than with FCS. Finally, human serum analysis revealed significantly higher levels of osteogenic components such as alkaline phosphatase and 25-Hydroxyvitamin D, consistent with the enhanced osteogenic effect. These results indicate that human serum used at the start of the culture offers an efficient replacement for continuous FCS treatment and could enable short-term exposure to patient-derived serum in the future. Full article

(This article belongs to the Special Issue Molecular and Tissue Engineering Approaches in Musculoskeletal Regenerative Medicine)

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16 pages, 4533 KiB

Open AccessArticle

Fibrin-Hyaluronic Acid Hydrogel (RegenoGel) with Fibroblast Growth Factor-18 for In Vitro 3D Culture of Human and Bovine Nucleus Pulposus Cells

by Sonja Häckel, Mona Zolfaghar, Jie Du, Sven Hoppe, Lorin M. Benneker, Nathalie Garstka, Marianna Peroglio, Mauro Alini, Sibylle Grad, Avner Yayon and Zhen Li

Int. J. Mol. Sci. 2019, 20(20), 5036; https://doi.org/10.3390/ijms20205036 - 11 Oct 2019

Cited by 19 | Viewed by 3743

Abstract

We investigated the effects of a fibrin-hyaluronic acid hydrogel (FBG–HA) and fibroblast growth factor 18 (FGF-18) for nucleus pulposus (NP) regeneration. Healthy bovine (n = 4) and human degenerated NP cells (n = 4) were cultured for 14 days in FBG-HA [...] Read more.

We investigated the effects of a fibrin-hyaluronic acid hydrogel (FBG–HA) and fibroblast growth factor 18 (FGF-18) for nucleus pulposus (NP) regeneration. Healthy bovine (n = 4) and human degenerated NP cells (n = 4) were cultured for 14 days in FBG-HA hydrogel with FGF-18 (∆51-mutant or wild-type) in the culture medium. Gene expression, DNA content, and glycosaminoglycan (GAG) synthesis were evaluated on day 7 and 14. Additionally, histology was performed. Human NP cells cultured in FBG-HA hydrogel showed an increase in collagen type II (COL2) and carbonic anhydrase XII (CA12) gene expression after 14 or 7 days of culture, respectively. GAG release into the conditioned medium increased over 14 days. Healthy bovine NP cells showed increased gene expression of ACAN from day 7 to day 14. Wild type FGF-18 up-regulated CA12 gene expression of human NP cells. Histology revealed an increase of proteoglycan deposition upon FGF-18 stimulation in bovine but not in human NP cells. The FBG-HA hydrogel had a positive modulatory effect on human degenerated NP cells. Under the tested conditions, no significant effect of FGF-18 was observed on cell proliferation or GAG synthesis in human NP cells. Full article

(This article belongs to the Special Issue Molecular and Tissue Engineering Approaches in Musculoskeletal Regenerative Medicine)

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11 pages, 5431 KiB

Open AccessArticle

In Vivo Bone Regeneration Induced by a Scaffold of Chitosan/Dicarboxylic Acid Seeded with Human Periodontal Ligament Cells

by Teerawat Sukpaita, Suwabun Chirachanchai, Pornchanok Suwattanachai, Vincent Everts, Atiphan Pimkhaokham and Ruchanee Salingcarnboriboon Ampornaramveth

Int. J. Mol. Sci. 2019, 20(19), 4883; https://doi.org/10.3390/ijms20194883 - 01 Oct 2019

Cited by 25 | Viewed by 3898

Abstract

Chitosan/dicarboxylic acid (CS/DA) scaffold has been developed as a bone tissue engineering material. This study evaluated a CS/DA scaffold with and without seeded primary human periodontal ligament cells (hPDLCs) in its capacity to regenerate bone in calvarial defects of mice. The osteogenic differentiation [...] Read more.

Chitosan/dicarboxylic acid (CS/DA) scaffold has been developed as a bone tissue engineering material. This study evaluated a CS/DA scaffold with and without seeded primary human periodontal ligament cells (hPDLCs) in its capacity to regenerate bone in calvarial defects of mice. The osteogenic differentiation of hPDLCs was analyzed by bone nodule formation and gene expression. In vivo bone regeneration was analyzed in mice calvarial defects. Eighteen mice were divided into 3 groups: one group with empty defects, one group with defects with CS/DA scaffold, and a group with defects with CS/DA scaffold and with hPDLCs. After 6 and 12 weeks, new bone formation was assessed using microcomputed tomography (Micro-CT) and histology. CS/DA scaffold significantly promoted in vitro osteoblast-related gene expression (RUNX2, OSX, COL1, ALP, and OPN) by hPDLCs. Micro-CT revealed that CS/DA scaffolds significantly promoted in vivo bone regeneration both after 6 and 12 weeks (p < 0.05). Histological examination confirmed these findings. New bone formation was observed in defects with CS/DA scaffold; being similar with and without hPDLCs. CS/DA scaffolds can be used as a bone regenerative material with good osteoinductive/osteoconductive properties. Full article

(This article belongs to the Special Issue Molecular and Tissue Engineering Approaches in Musculoskeletal Regenerative Medicine)

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26 pages, 9576 KiB

Open AccessArticle

Induction of Articular Chondrogenesis by Chitosan/Hyaluronic-Acid-Based Biomimetic Matrices Using Human Adipose-Derived Stem Cells

by Yijiang Huang, Daniel Seitz, Fabian König, Peter E. Müller, Volkmar Jansson and Roland M. Klar

Int. J. Mol. Sci. 2019, 20(18), 4487; https://doi.org/10.3390/ijms20184487 - 11 Sep 2019

Cited by 40 | Viewed by 4209

Abstract

Cartilage repair using tissue engineering is the most advanced clinical application in regenerative medicine, yet available solutions remain unsuccessful in reconstructing native cartilage in its proprietary form and function. Previous investigations have suggested that the combination of specific bioactive elements combined with a [...] Read more.

Cartilage repair using tissue engineering is the most advanced clinical application in regenerative medicine, yet available solutions remain unsuccessful in reconstructing native cartilage in its proprietary form and function. Previous investigations have suggested that the combination of specific bioactive elements combined with a natural polymer could generate carrier matrices that enhance activities of seeded stem cells and possibly induce the desired matrix formation. The present study sought to clarify this by assessing whether a chitosan-hyaluronic-acid-based biomimetic matrix in conjunction with adipose-derived stem cells could support articular hyaline cartilage formation in relation to a standard chitosan-based construct. By assessing cellular development, matrix formation, and key gene/protein expressions during in vitro cultivation utilizing quantitative gene and immunofluorescent assays, results showed that chitosan with hyaluronic acid provides a suitable environment that supports stem cell differentiation towards cartilage matrix producing chondrocytes. However, on the molecular gene expression level, it has become apparent that, without combinations of morphogens, in the chondrogenic medium, hyaluronic acid with chitosan has a very limited capacity to stimulate and maintain stem cells in an articular chondrogenic state, suggesting that cocktails of various growth factors are one of the key features to regenerate articular cartilage, clinically. Full article

(This article belongs to the Special Issue Molecular and Tissue Engineering Approaches in Musculoskeletal Regenerative Medicine)

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18 pages, 2023 KiB

Open AccessArticle

Supplementation with 45S5 Bioactive Glass Reduces In Vivo Resorption of the β-Tricalcium-Phosphate-Based Bone Substitute Material Vitoss

by Fabian Westhauser, Christopher Essers, Maria Karadjian, Bruno Reible, Gerhard Schmidmaier, Sébastien Hagmann and Arash Moghaddam

Int. J. Mol. Sci. 2019, 20(17), 4253; https://doi.org/10.3390/ijms20174253 - 30 Aug 2019

Cited by 13 | Viewed by 3173

Abstract

Compared to other materials such as 45S5 bioactive glass (BG), β-tricalcium phosphate (β-TCP)-based bone substitutes such as Vitoss show limited material-driven stimulation of osteogenesis and/or angiogenesis. The unfavorable degradation kinetics of β-TCP-based bone substitutes may result in an imbalance between resorption and osseous [...] Read more.

Compared to other materials such as 45S5 bioactive glass (BG), β-tricalcium phosphate (β-TCP)-based bone substitutes such as Vitoss show limited material-driven stimulation of osteogenesis and/or angiogenesis. The unfavorable degradation kinetics of β-TCP-based bone substitutes may result in an imbalance between resorption and osseous regeneration. Composite materials like Vitoss BA (Vitoss supplemented with 20 wt % 45S5-BG particles) might help to overcome these limitations. However, the influence of BG particles in Vitoss BA compared to unsupplemented Vitoss on osteogenesis, resorption behavior, and angiogenesis is not yet described. In this study, Vitoss and Vitoss BA scaffolds were seeded with human mesenchymal stromal cells before subcutaneous implantation in immunodeficient mice for 10 weeks. Scaffold resorption was monitored by micro-computed tomography, while osteoid formation and vascularization were assessed by histomorphometry and gene expression analysis. Whilst slightly more osteoid and improved angiogenesis were found in Vitoss BA, maturation of the osteoid was more advanced in Vitoss scaffolds. The volume of Vitoss implants decreased significantly, combined with a significantly increased presence of resorbing cells, whilst the volume remained stable in Vitoss BA scaffolds. Future studies should evaluate the interaction of 45S5-BG with resorbing cells and bone precursor cells in greater detail to improve the understanding and application of β-TCP/45S5-BG composite bone substitute materials. Full article

(This article belongs to the Special Issue Molecular and Tissue Engineering Approaches in Musculoskeletal Regenerative Medicine)

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15 pages, 6055 KiB

Open AccessArticle

Clumps of Mesenchymal Stem Cell/Extracellular Matrix Complexes Generated with Xeno-Free Conditions Facilitate Bone Regeneration via Direct and Indirect Osteogenesis

by Souta Motoike, Mikihito Kajiya, Nao Komatsu, Susumu Horikoshi, Tomoya Ogawa, Hisakatsu Sone, Shinji Matsuda, Kazuhisa Ouhara, Tomoyuki Iwata, Noriyoshi Mizuno, Tsuyoshi Fujita, Makoto Ikeya and Hidemi Kurihara

Int. J. Mol. Sci. 2019, 20(16), 3970; https://doi.org/10.3390/ijms20163970 - 15 Aug 2019

Cited by 21 | Viewed by 5435

Abstract

Three-dimensional clumps of mesenchymal stem cell (MSC)/extracellular matrix (ECM) complexes (C-MSCs) consist of cells and self-produced ECM. We demonstrated previously that C-MSCs can be transplanted into bone defect regions with no artificial scaffold to induce bone regeneration. To apply C-MSCs in a clinical [...] Read more.

Three-dimensional clumps of mesenchymal stem cell (MSC)/extracellular matrix (ECM) complexes (C-MSCs) consist of cells and self-produced ECM. We demonstrated previously that C-MSCs can be transplanted into bone defect regions with no artificial scaffold to induce bone regeneration. To apply C-MSCs in a clinical setting as a reliable bone regenerative therapy, the present study aimed to generate C-MSCs in xeno-free/serum-free conditions that can exert successful bone regenerative properties and to monitor interactions between grafted cells and host cells during bone healing processes. Human bone marrow-derived MSCs were cultured in xeno-free/serum-free medium. To obtain C-MSCs, confluent cells that had formed on the cellular sheet were scratched using a micropipette tip and then torn off. The sheet was rolled to make a round clump of cells. Then, C-MSCs were transplanted into an immunodeficient mouse calvarial defect model. Transplantation of C-MSCs induced bone regeneration in a time-dependent manner. Immunofluorescence staining showed that both donor human cells and host mice cells contributed to bone reconstruction. Decellularized C-MSCs implantation failed to induce bone regeneration, even though the host mice cells can infiltrate into the defect area. These findings suggested that C-MSCs generated in xeno-free/serum-free conditions can induce bone regeneration via direct and indirect osteogenesis. Full article

(This article belongs to the Special Issue Molecular and Tissue Engineering Approaches in Musculoskeletal Regenerative Medicine)

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20 pages, 9657 KiB

Open AccessArticle

Biological Response to Macroporous Chitosan-Agarose Bone Scaffolds Comprising Mg- and Zn-Doped Nano-Hydroxyapatite

by Paulina Kazimierczak, Joanna Kolmas and Agata Przekora

Int. J. Mol. Sci. 2019, 20(15), 3835; https://doi.org/10.3390/ijms20153835 - 06 Aug 2019

Cited by 35 | Viewed by 4940

Abstract

Modification of implantable scaffolds with magnesium and zinc for improvement of bone regeneration is a growing trend in the engineering of biomaterials. The aim of this study was to synthesize nano-hydroxyapatite substituted with magnesium (Mg²⁺) (HA-Mg) and zinc (Zn²⁺) [...] Read more.

Modification of implantable scaffolds with magnesium and zinc for improvement of bone regeneration is a growing trend in the engineering of biomaterials. The aim of this study was to synthesize nano-hydroxyapatite substituted with magnesium (Mg²⁺) (HA-Mg) and zinc (Zn²⁺) (HA-Zn) ions in order to fabricate chitosan-agarose-hydroxyapatite (HA) scaffolds (chit/aga/HA) with improved biocompatibility. Fabricated biomaterials containing Mg²⁺ or Zn²⁺ were tested using osteoblasts and mesenchymal stem cells to determine the effect of incorporated metal ions on cell adhesion, spreading, proliferation, and osteogenic differentiation. The study was conducted in direct contact with the scaffolds (cells were seeded onto the biomaterials) and using fluid extracts of the materials. It demonstrated that incorporation of Mg²⁺ ions into chit/aga/HA structure increased spreading of the osteoblasts, promoted cell proliferation on the scaffold surface, and enhanced osteocalcin production by mesenchymal stem cells. Although biomaterial containing Zn²⁺ did not improve cell proliferation, it did enhance type I collagen production by mesenchymal stem cells and extracellular matrix mineralization as compared to cells cultured in a polystyrene well. Nevertheless, scaffolds made of pure HA gave better results than material with Zn²⁺. Results of the experiments clearly showed that modification of the chit/aga/HA scaffold with Zn²⁺ did not have any positive impact on cell behavior, whereas, incorporation of Mg²⁺ ions into its structure may significantly improve biocompatibility of the resultant material, increasing its potential in biomedical applications. Full article

(This article belongs to the Special Issue Molecular and Tissue Engineering Approaches in Musculoskeletal Regenerative Medicine)

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18 pages, 4033 KiB

Open AccessArticle

In Vivo Response of Growth Plate to Biodegradable Mg-Ca-Zn Alloys Depending on the Surface Modification

by Mi Hyun Song, Won Joon Yoo, Tae-Joon Cho, Yong Koo Park, Wang-Jae Lee and In Ho Choi

Int. J. Mol. Sci. 2019, 20(15), 3761; https://doi.org/10.3390/ijms20153761 - 01 Aug 2019

Cited by 13 | Viewed by 2779

Abstract

Because Mg-Ca-Zn alloys are biodegradable and obviate secondary implant removal, they are especially beneficial for pediatric patients. We examined the degradation performance of Mg-Ca-Zn alloys depending on the surface modification and investigated the in vivo effects on the growth plate in a skeletally [...] Read more.

Because Mg-Ca-Zn alloys are biodegradable and obviate secondary implant removal, they are especially beneficial for pediatric patients. We examined the degradation performance of Mg-Ca-Zn alloys depending on the surface modification and investigated the in vivo effects on the growth plate in a skeletally immature rabbit model. Either plasma electrolyte oxidation (PEO)-coated (n = 18) or non-coated (n = 18) Mg-Ca-Zn alloy was inserted at the distal femoral physis. We measured the degradation performance and femoral segment lengths using micro-CT. In addition, we analyzed the histomorphometric and histopathologic characteristics of the growth plate. Although there were no acute, chronic inflammatory reactions in either group, they differed significantly in the tissue reactions to their degradation performance and physeal responses. Compared to non-coated alloys, PEO-coated alloys degraded significantly slowly with diminished hydrogen gas formation. Depending on the degradation rate, large bone bridge formation and premature physeal arrest occurred primarily in the non-coated group, whereas only a small-sized bone bridge formed in the PEO-coated group. This difference ultimately led to significant shortening of the femoral segment in the non-coated group. This study suggests that optimal degradation could be achieved with PEO-coated Mg-Ca-Zn alloys, making them promising and safe biodegradable materials with no growth plate damage. Full article

(This article belongs to the Special Issue Molecular and Tissue Engineering Approaches in Musculoskeletal Regenerative Medicine)

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22 pages, 6130 KiB

Open AccessArticle

Phenotypic Characterization of Bone Marrow Mononuclear Cells and Derived Stromal Cell Populations from Human Iliac Crest, Vertebral Body and Femoral Head

by Marietta Herrmann, Maria Hildebrand, Ursula Menzel, Niamh Fahy, Mauro Alini, Siegmund Lang, Lorin Benneker, Sophie Verrier, Martin J. Stoddart and Jennifer J. Bara

Int. J. Mol. Sci. 2019, 20(14), 3454; https://doi.org/10.3390/ijms20143454 - 14 Jul 2019

Cited by 30 | Viewed by 4742

Abstract

(1) In vitro, bone marrow-derived stromal cells (BMSCs) demonstrate inter-donor phenotypic variability, which presents challenges for the development of regenerative therapies. Here, we investigated whether the frequency of putative BMSC sub-populations within the freshly isolated mononuclear cell fraction of bone marrow is phenotypically [...] Read more.

(1) In vitro, bone marrow-derived stromal cells (BMSCs) demonstrate inter-donor phenotypic variability, which presents challenges for the development of regenerative therapies. Here, we investigated whether the frequency of putative BMSC sub-populations within the freshly isolated mononuclear cell fraction of bone marrow is phenotypically predictive for the in vitro derived stromal cell culture. (2) Vertebral body, iliac crest, and femoral head bone marrow were acquired from 33 patients (10 female and 23 male, age range 14–91). BMSC sub-populations were identified within freshly isolated mononuclear cell fractions based on cell-surface marker profiles. Stromal cells were expanded in monolayer on tissue culture plastic. Phenotypic assessment of in vitro derived cell cultures was performed by examining growth kinetics, chondrogenic, osteogenic, and adipogenic differentiation. (3) Gender, donor age, and anatomical site were neither predictive for the total yield nor the population doubling time of in vitro derived BMSC cultures. The abundance of freshly isolated progenitor sub-populations (CD45−CD34−CD73+, CD45−CD34−CD146+, NG2+CD146+) was not phenotypically predictive of derived stromal cell cultures in terms of growth kinetics nor plasticity. BMSCs derived from iliac crest and vertebral body bone marrow were more responsive to chondrogenic induction, forming superior cartilaginous tissue in vitro, compared to those isolated from femoral head. (4) The identification of discrete progenitor populations in bone marrow by current cell-surface marker profiling is not predictive for subsequently derived in vitro BMSC cultures. Overall, the iliac crest and the vertebral body offer a more reliable tissue source of stromal progenitor cells for cartilage repair strategies compared to femoral head. Full article

(This article belongs to the Special Issue Molecular and Tissue Engineering Approaches in Musculoskeletal Regenerative Medicine)

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17 pages, 4096 KiB

Open AccessArticle

Norepinephrine Inhibits Synovial Adipose Stem Cell Chondrogenesis via α2a-Adrenoceptor-Mediated ERK1/2 Activation

by Karima El Bagdadi, Frank Zaucke, Andrea Meurer, Rainer H. Straub and Zsuzsa Jenei-Lanzl

Int. J. Mol. Sci. 2019, 20(13), 3127; https://doi.org/10.3390/ijms20133127 - 26 Jun 2019

Cited by 12 | Viewed by 4654

Abstract

In recent years, first evidences emerged that sympathetic neurotransmitters influence osteoarthritis (OA) manifestation. Joint-resident stem cells might contribute to cartilage repair, however, their chondrogenic function is reduced. The neurotransmitter norepinephrine (NE) was detected in the synovial fluid of trauma and OA patients. Therefore, [...] Read more.

In recent years, first evidences emerged that sympathetic neurotransmitters influence osteoarthritis (OA) manifestation. Joint-resident stem cells might contribute to cartilage repair, however, their chondrogenic function is reduced. The neurotransmitter norepinephrine (NE) was detected in the synovial fluid of trauma and OA patients. Therefore, the aim of this study was to analyse how NE influences the chondrogenesis of synovial adipose tissue-derived stem cells (sASCs). sASCs were isolated from knee-OA patients synovia. After adrenoceptor (AR) expression analysis, proliferation and chondrogenic differentiation in presence of NE and/or α- and β-AR antagonist were investigated. Cell count, viability, chondrogenic and hypertophic gene expression, sulfated glycosaminoglycan (sGAG) and type II collagen content were determined. Key AR-dependent signaling (ERK1/2, PKA) was analyzed via western blot. sASC expressed α1A-, α1B-, α2A-, α2B-, α2C-, and β2-AR in monolayer and pellet culture. NE did not affect proliferation and viability, but 10⁻⁷ and 10⁻⁶ M NE significantly reduced sGAG and type II collagen content as well as ERK1/2 phosphorylation. These effects were fully reversed by yohimbine (α2-AR antagonist). Our study confirms the important role of NE in sASC chondrogenic function and provides new insights in OA pathophysiology. Future studies might help to develop novel therapeutic options targeting neuroendocrine pathways for OA treatment. Full article

(This article belongs to the Special Issue Molecular and Tissue Engineering Approaches in Musculoskeletal Regenerative Medicine)

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14 pages, 2155 KiB

Open AccessArticle

Therapeutic Effects of rAAV-Mediated Concomittant Gene Transfer and Overexpression of TGF-β and IGF-I on the Chondrogenesis of Human Bone-Marrow-Derived Mesenchymal Stem Cells

by Stephanie Morscheid, Ana Rey-Rico, Gertrud Schmitt, Henning Madry, Magali Cucchiarini and Jagadeesh Kumar Venkatesan

Int. J. Mol. Sci. 2019, 20(10), 2591; https://doi.org/10.3390/ijms20102591 - 27 May 2019

Cited by 9 | Viewed by 3771

Abstract

Application of chondroreparative gene vectors in cartilage defects is a powerful approach to directly stimulate the regenerative activities of bone-marrow-derived mesenchymal stem cells (MSCs) that repopulate such lesions. Here, we investigated the ability of combined recombinant adeno-associated virus (rAAV) vector-mediated delivery of the [...] Read more.

Application of chondroreparative gene vectors in cartilage defects is a powerful approach to directly stimulate the regenerative activities of bone-marrow-derived mesenchymal stem cells (MSCs) that repopulate such lesions. Here, we investigated the ability of combined recombinant adeno-associated virus (rAAV) vector-mediated delivery of the potent transforming growth factor beta (TGF-β) and insulin-like growth factor I (IGF-I) to enhance the processes of chondrogenic differentiation in human MSCs (hMSCs) relative to individual candidate treatments and to reporter (lacZ) gene condition. The rAAV-hTGF-β and rAAV-hIGF-I vectors were simultaneously provided to hMSC aggregate cultures (TGF-β/IGF-I condition) in chondrogenic medium over time (21 days) versus TGF-β/lacZ, IGF-I/lacZ, and lacZ treatments at equivalent vector doses. The cultures were then processed to monitor transgene (co)-overexpression, the levels of biological activities in the cells (cell proliferation, matrix synthesis), and the development of a chondrogenic versus osteogenic/hypertrophic phenotype. Effective, durable co-overexpression of TGF-β with IGF-I via rAAV enhanced the proliferative, anabolic, and chondrogenic activities in hMSCs versus lacZ treatment and reached levels that were higher than those achieved upon single candidate gene transfer, while osteogenic/hypertrophic differentiation was delayed over the period of time evaluated. These findings demonstrate the potential of manipulating multiple therapeutic rAAV vectors as a tool to directly target bone-marrow-derived MSCs in sites of focal cartilage defects and to locally enhance the endogenous processes of cartilage repair. Full article

(This article belongs to the Special Issue Molecular and Tissue Engineering Approaches in Musculoskeletal Regenerative Medicine)

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14 pages, 3918 KiB

Open AccessArticle

A Composite Chitosan-Reinforced Scaffold Fails to Provide Osteochondral Regeneration

by Alice Roffi, Elizaveta Kon, Francesco Perdisa, Milena Fini, Alessandro Di Martino, Annapaola Parrilli, Francesca Salamanna, Monica Sandri, Maria Sartori, Simone Sprio, Anna Tampieri, Maurilio Marcacci and Giuseppe Filardo

Int. J. Mol. Sci. 2019, 20(9), 2227; https://doi.org/10.3390/ijms20092227 - 07 May 2019

Cited by 19 | Viewed by 3759

Abstract

Several biomaterials have recently been developed to address the challenge of osteochondral regeneration. Among these, chitosan holds promises both for cartilage and bone healing. The aim of this in vivo study was to evaluate the regeneration potential of a novel hybrid magnesium-doped hydroxyapatite [...] Read more.

Several biomaterials have recently been developed to address the challenge of osteochondral regeneration. Among these, chitosan holds promises both for cartilage and bone healing. The aim of this in vivo study was to evaluate the regeneration potential of a novel hybrid magnesium-doped hydroxyapatite (MgHA), collagen, chitosan-based scaffold, which was tested in a sheep model to ascertain its osteochondral regenerative potential, and in a rabbit model to further evaluate its ability to regenerate bone tissue. Macroscopic, microtomography, histology, histomorphometry, and immunohistochemical analysis were performed. In the sheep model, all analyses did not show significant differences compared to untreated defects (p > 0.05), with no evidence of cartilage and subchondral bone regeneration. In the rabbit model, this bone scaffold provided less ability to enhance tissue healing compared with a commercial bone scaffold. Moreover, persistence of scaffold material and absence of integration with connective tissue around the scaffolds were observed. These results raised some concerns about the osteochondral use of this chitosan composite scaffold, especially for the bone layer. Further studies are needed to explore the best formulation of chitosan-reinforced composites for osteochondral treatment. Full article

(This article belongs to the Special Issue Molecular and Tissue Engineering Approaches in Musculoskeletal Regenerative Medicine)

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Review

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24 pages, 643 KiB

Open AccessReview

Usefulness of Mesenchymal Cell Lines for Bone and Cartilage Regeneration Research

by M. Piñeiro-Ramil, C. Sanjurjo-Rodríguez, R. Castro-Viñuelas, S. Rodríguez-Fernández, I.M. Fuentes-Boquete, F.J. Blanco and S.M. Díaz-Prado

Int. J. Mol. Sci. 2019, 20(24), 6286; https://doi.org/10.3390/ijms20246286 - 13 Dec 2019

Cited by 18 | Viewed by 3581

Abstract

The unavailability of sufficient numbers of human primary cells is a major roadblock for in vitro repair of bone and/or cartilage, and for performing disease modelling experiments. Immortalized mesenchymal stromal cells (iMSCs) may be employed as a research tool for avoiding these problems. [...] Read more.

The unavailability of sufficient numbers of human primary cells is a major roadblock for in vitro repair of bone and/or cartilage, and for performing disease modelling experiments. Immortalized mesenchymal stromal cells (iMSCs) may be employed as a research tool for avoiding these problems. The purpose of this review was to revise the available literature on the characteristics of the iMSC lines, paying special attention to the maintenance of the phenotype of the primary cells from which they were derived, and whether they are effectively useful for in vitro disease modeling and cell therapy purposes. This review was performed by searching on Web of Science, Scopus, and PubMed databases from 1 January 2015 to 30 September 2019. The keywords used were ALL = (mesenchymal AND (“cell line” OR immortal*) AND (cartilage OR chondrogenesis OR bone OR osteogenesis) AND human). Only original research studies in which a human iMSC line was employed for osteogenesis or chondrogenesis experiments were included. After describing the success of the immortalization protocol, we focused on the iMSCs maintenance of the parental phenotype and multipotency. According to the literature revised, it seems that the maintenance of these characteristics is not guaranteed by immortalization, and that careful selection and validation of clones with particular characteristics is necessary for taking advantage of the full potential of iMSC to be employed in bone and cartilage-related research. Full article

(This article belongs to the Special Issue Molecular and Tissue Engineering Approaches in Musculoskeletal Regenerative Medicine)

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26 pages, 1988 KiB

Open AccessReview

Current and Future Concepts for the Treatment of Impaired Fracture Healing

by Carsten W. Schlickewei, Holger Kleinertz, Darius M. Thiesen, Konrad Mader, Matthias Priemel, Karl-Heinz Frosch and Johannes Keller

Int. J. Mol. Sci. 2019, 20(22), 5805; https://doi.org/10.3390/ijms20225805 - 19 Nov 2019

Cited by 48 | Viewed by 10506

Abstract

Bone regeneration represents a complex process, of which basic biologic principles have been evolutionarily conserved over a broad range of different species. Bone represents one of few tissues that can heal without forming a fibrous scar and, as such, resembles a unique form [...] Read more.

Bone regeneration represents a complex process, of which basic biologic principles have been evolutionarily conserved over a broad range of different species. Bone represents one of few tissues that can heal without forming a fibrous scar and, as such, resembles a unique form of tissue regeneration. Despite a tremendous improvement in surgical techniques in the past decades, impaired bone regeneration including non-unions still affect a significant number of patients with fractures. As impaired bone regeneration is associated with high socio-economic implications, it is an essential clinical need to gain a full understanding of the pathophysiology and identify novel treatment approaches. This review focuses on the clinical implications of impaired bone regeneration, including currently available treatment options. Moreover, recent advances in the understanding of fracture healing are discussed, which have resulted in the identification and development of novel therapeutic approaches for affected patients. Full article

(This article belongs to the Special Issue Molecular and Tissue Engineering Approaches in Musculoskeletal Regenerative Medicine)

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15 pages, 1891 KiB

Open AccessReview

Intervertebral Disc Nucleus Repair: Hype or Hope?

by Gauri Tendulkar, Tao Chen, Sabrina Ehnert, Hans-Peter Kaps and Andreas K Nüssler

Int. J. Mol. Sci. 2019, 20(15), 3622; https://doi.org/10.3390/ijms20153622 - 24 Jul 2019

Cited by 51 | Viewed by 9865

Abstract

Chronic back pain is a common disability, which is often accredited to intervertebral disc degeneration. Gold standard interventions such as spinal fusion, which are mainly designed to mechanically seal the defect, frequently fail to restore the native biomechanics. Moreover, artificial implants have limited [...] Read more.

Chronic back pain is a common disability, which is often accredited to intervertebral disc degeneration. Gold standard interventions such as spinal fusion, which are mainly designed to mechanically seal the defect, frequently fail to restore the native biomechanics. Moreover, artificial implants have limited success as a repair strategy, as they do not alter the underlying disease and fail to promote tissue integration and subsequent native biomechanics. The reported high rates of spinal fusion and artificial disc implant failure have pushed intervertebral disc degeneration research in recent years towards repair strategies. Intervertebral disc repair utilizing principles of tissue engineering should theoretically be successful, overcoming the inadequacies of artificial implants. For instance, advances in the development of scaffolds aided with cells and growth factors have opened up new possibilities for repair strategies. However, none has reached the stage of clinical trials in humans. In this review, we describe the hitches encountered in the musculoskeletal field and summarize recent advances in designing tissue-engineered constructs for promoting nucleus pulposus repair. Additionally, the review focuses on the effect of biomaterial aided with cells and growth factors on achieving effective functional reparative potency, highlighting the ways to enhance the efficacy of these treatments. Full article

(This article belongs to the Special Issue Molecular and Tissue Engineering Approaches in Musculoskeletal Regenerative Medicine)

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