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Molecular and Tissue Engineering Approaches in Musculoskeletal Regenerative Medicine 2.0

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A special issue of International Journal of Molecular Sciences (ISSN 1422-0067). This special issue belongs to the section "Molecular Pathology, Diagnostics, and Therapeutics".

Deadline for manuscript submissions: closed (20 October 2020) | Viewed by 33753

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Prof. Dr. Magali Cucchiarini

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Center of Experimental Orthopaedics, Saarland University Medical Center, Kirrbergerstr. Bldg 37, D-66421 Homburg, Saar, Germany
Interests: molecular gene therapy; human regenerative medicine; tissue engineering; musculoskeletal research; stem cells
Special Issues, Collections and Topics in MDPI journals

Prof. Dr. Henning Madry

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Center of Experimental Orthopaedics, Saarland University Medical Center, Kirrbergerstr. Bldg 37, D-66421 Homburg, Saar, Germany
Interests: human musculoskeletal disorders; clinical orthopaedics; animal models; human regenerative medicine
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

This Special Issue is a continuation of our previous Special Issue "Molecular and Tissue Engineering Approaches in Musculoskeletal Regenerative Medicine" (https://www.mdpi.com/journal/ijms/special_issues/musculoskeletal_reg).

Injuries affecting the various tissues of the musculoskeletal system (articular cartilage, bone, meniscus, and tendons/ligaments) do not fully heal by themselves due to a limited or unsatisfactory ability of these tissues for spontaneous repair. While a number of clinical options are available to address such problems, none are capable to reproduce the native tissue structures and original functions in patients, showing the vital need for novel alternatives that may improve the current therapies by stimulating the reparative processes in sites of injury. In this regard, a number of molecular options may be envisaged, alone or in combination, based on the application of regenerative (differentiated or progenitor) cells, candidate genes, and biomaterials adapted for each type of tissue and disease. The goal of this Special Issue is to offer an overview of the most advanced procedures that may be used as tools to improve the healing of musculoskeletal disorders in future translational approaches.

Prof. Dr. Magali Cucchiarini
Prof. Dr. Henning Madry
Guest Editors

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Published Papers (10 papers)

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Research

18 pages, 27545 KiB

Open AccessArticle

Evaluation of Allogeneic Bone-Marrow-Derived and Umbilical Cord Blood-Derived Mesenchymal Stem Cells to Prevent the Development of Osteoarthritis in An Equine Model

by Lélia Bertoni, Sandrine Jacquet-Guibon, Thomas Branly, Mélanie Desancé, Florence Legendre, Martine Melin, Pascaline Rivory, Daniel-Jean Hartmann, Amandine Schmutz, Jean-Marie Denoix, Magali Demoor, Fabrice Audigié and Philippe Galéra

Int. J. Mol. Sci. 2021, 22(5), 2499; https://doi.org/10.3390/ijms22052499 - 2 Mar 2021

Cited by 15 | Viewed by 2813

Abstract

Osteoarthritis (OA) is a significant cause of pain in both humans and horses with a high socio-economic impact. The horse is recognized as a pertinent model for human OA. In both species, regenerative therapy with allogeneic mesenchymal stem cells (MSCs) appears to be a promising treatment but, to date, no in vivo studies have attempted to compare the effects of different cell sources on the same individuals. The objective of this study is to evaluate the ability of a single blinded intra-articular injection of allogeneic bone-marrow (BM) derived MSCs and umbilical cord blood (UCB) derived MSC to limit the development of OA-associated pathological changes compared to placebo in a post-traumatic OA model applied to all four fetlock joints of eight horses. The effect of the tissue source (BM vs. UCB) is also assessed on the same individuals. Observations were carried out using clinical, radiographic, ultrasonographic, and magnetic resonance imaging methods as well as biochemical analysis of synovial fluid and postmortem microscopic and macroscopic evaluations of the joints until Week 12. A significant reduction in the progression of OA-associated changes measured with imaging techniques, especially radiography, was observed after injection of bone-marrow derived mesenchymal stem cells (BM-MSCs) compared to contralateral placebo injections. These results indicate that allogeneic BM-MSCs are a promising treatment for OA in horses and reinforce the importance of continuing research to validate these results and find innovative strategies that will optimize the therapeutic potential of these cells. However, they should be considered with caution given the low number of units per group. Full article

(This article belongs to the Special Issue Molecular and Tissue Engineering Approaches in Musculoskeletal Regenerative Medicine 2.0)

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21 pages, 2831 KiB

Open AccessArticle

Stable Reference Genes for qPCR Analysis in BM-MSCs Undergoing Osteogenic Differentiation within 3D Hyaluronan-Based Hydrogels

by Johannes Hasler, Luan Phelipe Hatt, Martin James Stoddart and Angela Rita Armiento

Int. J. Mol. Sci. 2020, 21(23), 9195; https://doi.org/10.3390/ijms21239195 - 2 Dec 2020

Cited by 5 | Viewed by 2443

Abstract

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) enables the monitoring of changes in cell phenotype via the high-throughput screening of numerous genes. RT-qPCR is a fundamental approach in numerous research fields, including biomaterials, yet little attention has been given to the potential impact of 3D versus monolayer (2D) cell culture and to the requirement for a constant validation of the multiple steps of gene expression analysis. The aim of this study is to use high-quality RNA to identify the most suitable reference genes for RT-qPCR analysis during the osteogenic differentiation of human bone marrow mesenchymal stem/stromal cells (BM-MSCs). BM-MSCs are cultured under osteogenic conditions for 28 days in 2D or within hyaluronic acid hydrogels (3D). RNA is subject to quality controls and is then used to identify the most stable reference genes using geNorm, NormFinder, and the ∆Cq method. The effect of the reverse transcriptase is investigated, as well as the expression of osteogenic-related markers. This study shows marked differences in the stability of reference genes between 2D (RPLP0/GAPDH) and 3D (OAZ1/PPIA) culture, suggesting that it is critical to choose appropriate reference genes for 3D osteogenic cell cultures. Thus, a thorough validation under specific experimental settings is essential to obtain meaningful gene expression results. Full article

(This article belongs to the Special Issue Molecular and Tissue Engineering Approaches in Musculoskeletal Regenerative Medicine 2.0)

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15 pages, 3045 KiB

Open AccessArticle

The Transcription Factor HAND1 Is Involved in Cortical Bone Mass through the Regulation of Collagen Expression

by Noriko Funato, Yuki Taga, Lindsay E. Laurie, Chisa Tometsuka, Masashi Kusubata and Kiyoko Ogawa-Goto

Int. J. Mol. Sci. 2020, 21(22), 8638; https://doi.org/10.3390/ijms21228638 - 16 Nov 2020

Cited by 5 | Viewed by 2303

Abstract

Temporal and/or spatial alteration of collagen family gene expression results in bone defects. However, how collagen expression controls bone size remains largely unknown. The basic helix-loop-helix transcription factor HAND1 is expressed in developing long bones and is involved in their morphogenesis. To understand the functional role of HAND1 and collagen in the postnatal development of long bones, we overexpressed Hand1 in the osteochondroprogenitors of model mice and found that the bone volumes of cortical bones decreased in Hand1^Tg/+;Twist2-Cre mice. Continuous Hand1 expression downregulated the gene expression of type I, V, and XI collagen in the diaphyses of long bones and was associated with decreased expression of Runx2 and Sp7/Osterix, encoding transcription factors involved in the transactivation of fibril-forming collagen genes. Members of the microRNA-196 family, which target the 3′ untranslated regions of COL1A1 and COL1A2, were significantly upregulated in Hand1^Tg/+;Twist2-Cre mice. Mass spectrometry revealed that the expression ratios of alpha 1(XI), alpha 2(XI), and alpha 2(V) in the diaphysis increased during postnatal development in wild-type mice, which was delayed in Hand1^Tg/+;Twist2-Cre mice. Our results demonstrate that HAND1 regulates bone size and morphology through osteochondroprogenitors, at least partially by suppressing postnatal expression of collagen fibrils in the cortical bones. Full article

(This article belongs to the Special Issue Molecular and Tissue Engineering Approaches in Musculoskeletal Regenerative Medicine 2.0)

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17 pages, 1795 KiB

Open AccessArticle

Compression Bioreactor-Based Mechanical Loading Induces Mobilization of Human Bone Marrow-Derived Mesenchymal Stromal Cells into Collagen Scaffolds In Vitro

by Carolina Gamez, Barbara Schneider-Wald, Karen Bieback, Andy Schuette, Sylvia Büttner, Mathias Hafner, Norbert Gretz and Markus L. Schwarz

Int. J. Mol. Sci. 2020, 21(21), 8249; https://doi.org/10.3390/ijms21218249 - 4 Nov 2020

Cited by 3 | Viewed by 2539

Abstract

Articular cartilage (AC) is an avascular tissue composed of scattered chondrocytes embedded in a dense extracellular matrix, in which nourishment takes place via the synovial fluid at the surface. AC has a limited intrinsic healing capacity, and thus mainly surgical techniques have been used to relieve pain and improve function. Approaches to promote regeneration remain challenging. The microfracture (MF) approach targets the bone marrow (BM) as a source of factors and progenitor cells to heal chondral defects in situ by opening small holes in the subchondral bone. However, the original function of AC is not obtained yet. We hypothesize that mechanical stimulation can mobilize mesenchymal stromal cells (MSCs) from BM reservoirs upon MF of the subchondral bone. Thus, the aim of this study was to compare the counts of mobilized human BM-MSCs (hBM-MSCs) in alginate-laminin (alginate-Ln) or collagen-I (col-I) scaffolds upon intermittent mechanical loading. The mechanical set up within an established bioreactor consisted of 10% strain, 0.3 Hz, breaks of 10 s every 180 cycles for 24 h. Contrary to previous findings using porcine MSCs, no significant cell count was found for hBM-MSCs into alginate-Ln scaffolds upon mechanical stimulation (8 ± 5 viable cells/mm³ for loaded and 4 ± 2 viable cells/mm³ for unloaded alginate-Ln scaffolds). However, intermittent mechanical stimulation induced the mobilization of hBM-MSCs into col-I scaffolds 10-fold compared to the unloaded col-I controls (245 ± 42 viable cells/mm³ vs. 22 ± 6 viable cells/mm³, respectively; p-value < 0.0001). Cells that mobilized into the scaffolds by mechanical loading did not show morphological changes. This study confirmed that hBM-MSCs can be mobilized in vitro from a reservoir toward col-I but not alginate-Ln scaffolds upon intermittent mechanical loading, against gravity. Full article

(This article belongs to the Special Issue Molecular and Tissue Engineering Approaches in Musculoskeletal Regenerative Medicine 2.0)

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14 pages, 4692 KiB

Open AccessArticle

Orthotopic Bone Formation by Streamlined Engineering and Devitalization of Human Hypertrophic Cartilage

by Sébastien Pigeot, Paul Emile Bourgine, Jaquiery Claude, Celeste Scotti, Adam Papadimitropoulos, Atanas Todorov, Christian Epple, Giuseppe M. Peretti and Ivan Martin

Int. J. Mol. Sci. 2020, 21(19), 7233; https://doi.org/10.3390/ijms21197233 - 30 Sep 2020

Cited by 8 | Viewed by 2853

Abstract

Most bones of the human body form and heal through endochondral ossification, whereby hypertrophic cartilage (HyC) is formed and subsequently remodeled into bone. We previously demonstrated that HyC can be engineered from human mesenchymal stromal cells (hMSC), and subsequently devitalized by apoptosis induction. The resulting extracellular matrix (ECM) tissue retained osteoinductive properties, leading to ectopic bone formation. In this study, we aimed at engineering and devitalizing upscaled quantities of HyC ECM within a perfusion bioreactor, followed by in vivo assessment in an orthotopic bone repair model. We hypothesized that the devitalized HyC ECM would outperform a clinical product currently used for bone reconstructive surgery. Human MSC were genetically engineered with a gene cassette enabling apoptosis induction upon addition of an adjuvant. Engineered hMSC were seeded, differentiated, and devitalized within a perfusion bioreactor. The resulting HyC ECM was subsequently implanted in a 10-mm rabbit calvarial defect model, with processed human bone (Maxgraft^®) as control. Human MSC cultured in the perfusion bioreactor generated a homogenous HyC ECM and were efficiently induced towards apoptosis. Following six weeks of in vivo implantation, microcomputed tomography and histological analyses of the defects revealed an increased bone formation in the defects filled with HyC ECM as compared to Maxgraft^®. This work demonstrates the suitability of engineered devitalized HyC ECM as a bone substitute material, with a performance superior to a state-of-the-art commercial graft. Streamlined generation of the devitalized tissue transplant within a perfusion bioreactor is relevant towards standardized and automated manufacturing of a clinical product. Full article

(This article belongs to the Special Issue Molecular and Tissue Engineering Approaches in Musculoskeletal Regenerative Medicine 2.0)

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19 pages, 6891 KiB

Open AccessArticle

Differential Production of Cartilage ECM in 3D Agarose Constructs by Equine Articular Cartilage Progenitor Cells and Mesenchymal Stromal Cells

by Stefanie Schmidt, Florencia Abinzano, Anneloes Mensinga, Jörg Teßmar, Jürgen Groll, Jos Malda, Riccardo Levato and Torsten Blunk

Int. J. Mol. Sci. 2020, 21(19), 7071; https://doi.org/10.3390/ijms21197071 - 25 Sep 2020

Cited by 10 | Viewed by 3727

Abstract

Identification of articular cartilage progenitor cells (ACPCs) has opened up new opportunities for cartilage repair. These cells may be used as alternatives for or in combination with mesenchymal stromal cells (MSCs) in cartilage engineering. However, their potential needs to be further investigated, since only a few studies have compared ACPCs and MSCs when cultured in hydrogels. Therefore, in this study, we compared chondrogenic differentiation of equine ACPCs and MSCs in agarose constructs as monocultures and as zonally layered co-cultures under both normoxic and hypoxic conditions. ACPCs and MSCs exhibited distinctly differential production of the cartilaginous extracellular matrix (ECM). For ACPC constructs, markedly higher glycosaminoglycan (GAG) contents were determined by histological and quantitative biochemical evaluation, both in normoxia and hypoxia. Differential GAG production was also reflected in layered co-culture constructs. For both cell types, similar staining for type II collagen was detected. However, distinctly weaker staining for undesired type I collagen was observed in the ACPC constructs. For ACPCs, only very low alkaline phosphatase (ALP) activity, a marker of terminal differentiation, was determined, in stark contrast to what was found for MSCs. This study underscores the potential of ACPCs as a promising cell source for cartilage engineering. Full article

(This article belongs to the Special Issue Molecular and Tissue Engineering Approaches in Musculoskeletal Regenerative Medicine 2.0)

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19 pages, 4915 KiB

Open AccessArticle

GLI1 and AXIN2 Are Distinctive Markers of Human Calvarial Mesenchymal Stromal Cells in Nonsyndromic Craniosynostosis

by Lorena Di Pietro, Marta Barba, Chiara Prampolini, Sabrina Ceccariglia, Paolo Frassanito, Alessia Vita, Enrico Guadagni, Davide Bonvissuto, Luca Massimi, Gianpiero Tamburrini, Ornella Parolini and Wanda Lattanzi

Int. J. Mol. Sci. 2020, 21(12), 4356; https://doi.org/10.3390/ijms21124356 - 19 Jun 2020

Cited by 21 | Viewed by 5004

Abstract

All skeletal bones house osteogenic stem cell niches, in which mesenchymal stromal cells (MSC) provide progenitors for tissue growth and regeneration. They have been widely studied in long bones formed through endochondral ossification. Limited information is available on the composition of the osteogenic niche in flat bones (i.e., skull vault bones) that develop through direct membranous ossification. Craniosynostosis (CS) is a congenital craniofacial defect due to the excessive and premature ossification of skull vault sutures. This study aimed at analysing the expression of GLI1, AXIN2 and THY1 in the context of the human skull vault, using nonsyndromic forms of CS (NCS) as a model to test their functional implication in the aberrant osteogenic process. The expression of selected markers was studied in NCS patients’ calvarial bone specimens, to assess the in vivo location of cells, and in MSC isolated thereof. The marker expression profile was analysed during in vitro osteogenic differentiation to validate the functional implication. Our results show that GLI1 and AXIN2 are expressed in periosteal and endosteal locations within the osteogenic niche of human calvarial bones. Their expression is higher in MSC isolated from calvarial bones than in those isolated from long bones and tends to decrease upon osteogenic commitment and differentiation. In particular, AXIN2 expression was lower in cells isolated from prematurely fused sutures than in those derived from patent sutures of NCS patients. This suggests that AXIN2 could reasonably represent a marker for the stem cell population that undergoes depletion during the premature ossification process occurring in CS. Full article

(This article belongs to the Special Issue Molecular and Tissue Engineering Approaches in Musculoskeletal Regenerative Medicine 2.0)

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16 pages, 3085 KiB

Open AccessArticle

Chondrogenic Potential of Pellet Culture Compared to High-Density Culture on a Bacterial Cellulose Hydrogel

by Nele Pascale Grigull, Julia Isabelle Redeker, Bärbel Schmitt, Maximilian Michael Saller, Veronika Schönitzer and Susanne Mayer-Wagner

Int. J. Mol. Sci. 2020, 21(8), 2785; https://doi.org/10.3390/ijms21082785 - 16 Apr 2020

Cited by 14 | Viewed by 3284

Abstract

Cell-based approaches of cartilage lesions use different culture systems to obtain optimal cell quality. Pellet cultures with high cellular density (HD) are the gold standard to keep chondrocytes in a differentiated stage. Bacterial cellulose (BC) hydrogel is discussed to prevent cellular aging and dedifferentiation. The hypothesis of this study was that HD culture on BC hydrogel (HD hydrogel) might reach the chondrogenic potential of pellet culture (pellet). Human articular osteoarthritic (OA) and non-osteoarthritic (non-OA) chondrocytes were cultured for seven days within pellets and compared to HD hydrogel and HD polystyrene. Gene expression analysis and histological assessment were performed. We observed no significant change of COL2A1 expression by the culture system (pellet, HD hydrogel and HD polystyrene) but a significant change of COL2A1/COL1A1-ratio, with the highest ratio in pellets. Chondrocytes on HD hydrogel showed an elevated expression of MMP13 and on polystyrene an increased expression of COL1A1 and MMP13. The patterns of gene expression changes observed in OA and non-OA chondrocytes in reaction to the different culture systems were similar in those two cell groups. Pellet cultures moreover formed a histomorphologically superior neocartilage. Concluding, human chondrocytes kept the potential to express COL2A1 in all HD culture systems. However, pellets excelled in a higher COL2A1/COL1A1-ratio, a higher extracellular matrix deposit and in not developing degeneration and dedifferentiation markers. This underlines the superiority of pellet culture in maintaining the chondrogenic potential of human chondrocytes in vitro. Full article

(This article belongs to the Special Issue Molecular and Tissue Engineering Approaches in Musculoskeletal Regenerative Medicine 2.0)

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18 pages, 9040 KiB

Open AccessArticle

Intramuscular Injection of Combined Calf Blood Compound (CFC) and Homeopathic Drug Tr14 Accelerates Muscle Regeneration In Vivo

by Patrick Belikan, Lisa Nauth, Lars-Christopher Färber, Frédéric Abel, Eva Langendorf, Philipp Drees, Pol Maria Rommens, Ulrike Ritz and Stefan G. Mattyasovszky

Int. J. Mol. Sci. 2020, 21(6), 2112; https://doi.org/10.3390/ijms21062112 - 19 Mar 2020

Cited by 5 | Viewed by 3280

Abstract

Skeletal muscle injuries in competitive sports cause lengthy absences of athletes from tournaments. This is of tremendous competitive and economic relevance for both the athletes and their respective clubs. Therapy for structural muscle lesions aims to promote regeneration and fast-track return-to-play. A common clinical treatment strategy for muscle injuries is the intramuscular injection of calf blood compound and the homeopathic drug, Tr14. Although the combination of these two agents was reported to reduce recovery time, the regulatory mechanism whereby this occurs remains unknown. In this in vivo study, we selected a rat model of mechanical muscle injury to investigate the effect of this combination therapy on muscle regeneration. Gene expression analysis and histological images revealed that this combined intramuscular injection for muscle lesions can enhance the expression of pro-myogenic genes and proteins and accelerate muscle regeneration. These findings are novel and depict the positive effects of calf blood compound and the homeopathic drug, Tr14, which are utilized in the field of Sports medicine. Full article

(This article belongs to the Special Issue Molecular and Tissue Engineering Approaches in Musculoskeletal Regenerative Medicine 2.0)

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18 pages, 6610 KiB

Open AccessArticle

Divergent Regulation of Myotube Formation and Gene Expression by E2 and EPA during In-Vitro Differentiation of C2C12 Myoblasts

by Orly Lacham-Kaplan, Donny M. Camera and John A. Hawley

Int. J. Mol. Sci. 2020, 21(3), 745; https://doi.org/10.3390/ijms21030745 - 23 Jan 2020

Cited by 9 | Viewed by 4747

Abstract

Estrogen (E2) and polyunsaturated fatty acids (n-3PUFA) supplements independently support general wellbeing and enhance muscle regeneration in-vivo and myotube formation in-vitro. However, the combined effect of E2 and n-3PUFA on myoblast differentiation is not known. The purpose of the study was to identify whether E2 and n-3PUFA possess a synergistic effect on in-vitro myogenesis. Mouse C2C12 myoblasts, a reliable model to reiterate myogenic events in-vitro, were treated with 10nM E2 and 50μM eicosapentaenoic acid (EPA) independently or combined, for 0–24 h or 0–120 h during differentiation. Immunofluorescence, targeted qPCR and next generation sequencing (NGS) were used to characterize morphological changes and differential expression of key genes involved in the regulation of myogenesis and muscle function pathways. E2 increased estrogen receptor α (Erα) and the expression of the mitogen-activated protein kinase 11 (Mapk11) within 1 h of treatment and improved myoblast differentiation and myotube formation. A significant reduction (p < 0.001) in myotube formation and in the expression of myogenic regulatory factors Mrfs (MyoD, Myog and Myh1) and the myoblast fusion related gene, Tmem8c, was observed in the presence of EPA and the combined E2/EPA treatment. Additionally, EPA treatment at 48 h of differentiation inhibited the majority of genes associated with the myogenic and striated muscle contraction pathways. In conclusion, EPA and E2 had no synergistic effect on myotube formation in-vitro. Independently, EPA inhibited myoblast differentiation and overrides the stimulatory effect of E2 when used in combination with E2. Full article

(This article belongs to the Special Issue Molecular and Tissue Engineering Approaches in Musculoskeletal Regenerative Medicine 2.0)

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