Analysis of the Dynamics of Bone Formation, Effect of Cell Seeding Density, and Potential of Allogeneic Cells in Cell-Based Bone Tissue Engineering in Goats

The efficient seeding of cells into porous scaffolds is important in bone tissue engineering techniques. To enhance efficiency, we modified the previously reported cell seeding techniques using low-pressure conditions. In this study, the effects of low pressure on bone marrow-derived stromal cells (BMSCs) of rats and the usefulness of the modified technique were assessed. There was no significant difference found in the proliferative and osteogenic capabilities among various low-pressure (50–760 mmHg, 1–10 min) conditions. To analyze the efficacies of the cell seeding techniques, BMSCs suspended in the plasma of rats were seeded into porous β-tricalcium phosphate (β-TCP) blocks by the following three procedures: 1) spontaneous penetration of cell suspension under atmospheric pressure (SP); 2) spontaneous penetration and subsequent low pressure treatment (SPSL), the conventional technique; and 3) spontaneous penetration under low pressure conditions (SPUL), the modified technique. Subsequently, these BMSCs/β-TCP composites were used for the analysis of cell seeding efficiency or in vivo bone formation capability. Both the number of BMSCs seeded into β-TCP blocks and the amount of bone formation of the SPUL group were significantly higher than those of the other groups. The SPUL method with a simple technique permits high cell seeding efficiency and is useful for bone tissue engineering using BMSCs and porous scaffolds.

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A novel gelatin/chitooligosaccharide/demineralized bone matrix composite scaffold and periosteum-derived mesenchymal stem cells for bone tissue engineering

Biomaterials Research ◽

10.1186/s40824-021-00220-y ◽

2021 ◽

Vol 25 (1) ◽

Author(s):

Thakoon Thitiset ◽

Siriporn Damrongsakkul ◽

Supansa Yodmuang ◽

Wilairat Leeanansaksiri ◽

Jirun Apinun ◽

...

Keyword(s):

Tissue Engineering ◽

Stem Cells ◽

Mesenchymal Stem Cells ◽

Bone Formation ◽

Bone Tissue Engineering ◽

Bone Tissue ◽

Demineralized Bone Matrix ◽

Bone Matrix ◽

Alp Activity

Abstract Background A novel biodegradable scaffold including gelatin (G), chitooligosaccharide (COS), and demineralized bone matrix (DBM) could play a significant part in bone tissue engineering. The present study aimed to investigate the biological characteristics of composite scaffolds in combination of G, COS, and DBM for in vitro cell culture and in vivo animal bioassays. Methods Three-dimensional scaffolds from the mixture of G, COS, and DBM were fabricated into 3 groups, namely, G, GC, and GCD using a lyophilization technique. The scaffolds were cultured with mesenchymal stem cells (MSCs) for 4 weeks to determine biological responses such as cell attachment and cell proliferation, alkaline phosphatase (ALP) activity, calcium deposition, cell morphology, and cell surface elemental composition. For the in vivo bioassay, G, GC, and GCD, acellular scaffolds were implanted subcutaneously in 8-week-old male Wistar rats for 4 weeks and 8 weeks. The explants were assessed for new bone formation using hematoxylin and eosin (H&E) staining and von Kossa staining. Results The MSCs could attach and proliferate on all three groups of scaffolds. Interestingly, the ALP activity of MSCs reached the greatest value on day 7 after cultured on the scaffolds, whereas the calcium assay displayed the highest level of calcium in MSCs on day 28. Furthermore, weight percentages of calcium and phosphorus on the surface of MSCs after cultivation on the GCD scaffolds increased when compared to those on other scaffolds. The scanning electron microscopy images showed that MSCs attached and proliferated on the scaffold surface thoroughly over the cultivation time. Mineral crystal aggregation was evident in GC and greatly in GCD scaffolds. H&E staining illustrated that G, GC, and GCD scaffolds displayed osteoid after 4 weeks of implantation and von Kossa staining confirmed the mineralization at 8 weeks in G, GC, and GCD scaffolds. Conclusion The MSCs cultured in GCD scaffolds revealed greater osteogenic differentiation than those cultured in G and GC scaffolds. Additionally, the G, GC, and GCD scaffolds could promote in vivo ectopic bone formation in rat model. The GCD scaffolds exhibited maximum osteoinductive capability compared with others and may be potentially used for bone regeneration.

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Protocol of Co-Culture of Human Osteoblasts and Osteoclasts to Test Biomaterials for Bone Tissue Engineering

Methods and Protocols ◽

10.3390/mps5010008 ◽

2022 ◽

Vol 5 (1) ◽

pp. 8

Author(s):

Giorgia Borciani ◽

Giorgia Montalbano ◽

Nicola Baldini ◽

Chiara Vitale-Brovarone ◽

Gabriela Ciapetti

Keyword(s):

Tissue Engineering ◽

Bone Tissue Engineering ◽

Bone Tissue ◽

Bone Cells ◽

Bone Cell ◽

Seeding Density ◽

Bone Homeostasis ◽

Bone Microenvironment

New biomaterials and scaffolds for bone tissue engineering (BTE) applications require to be tested in a bone microenvironment reliable model. On this assumption, the in vitro laboratory protocols with bone cells represent worthy experimental systems improving our knowledge about bone homeostasis, reducing the costs of experimentation. To this day, several models of the bone microenvironment are reported in the literature, but few delineate a protocol for testing new biomaterials using bone cells. Herein we propose a clear protocol to set up an indirect co-culture system of human-derived osteoblasts and osteoclast precursors, providing well-defined criteria such as the cell seeding density, cell:cell ratio, the culture medium, and the proofs of differentiation. The material to be tested may be easily introduced in the system and the cell response analyzed. The physical separation of osteoblasts and osteoclasts allows distinguishing the effects of the material onto the two cell types and to evaluate the correlation between material and cell behavior, cell morphology, and adhesion. The whole protocol requires about 4 to 6 weeks with an intermediate level of expertise. The system is an in vitro model of the bone remodeling system useful in testing innovative materials for bone regeneration, and potentially exploitable in different application fields. The use of human primary cells represents a close replica of the bone cell cooperation in vivo and may be employed as a feasible system to test materials and scaffolds for bone substitution and regeneration.

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Comparison of the Anabolic Effects of Reported Osteogenic Compounds on Human Mesenchymal Progenitor-Derived Osteoblasts

Bioengineering ◽

10.3390/bioengineering7010012 ◽

2020 ◽

Vol 7 (1) ◽

pp. 12 ◽

Cited By ~ 3

Author(s):

Robert Owen ◽

Hossein Bahmaee ◽

Frederik Claeyssens ◽

Gwendolen C. Reilly

Keyword(s):

Tissue Engineering ◽

Bone Formation ◽

Bone Tissue Engineering ◽

Bone Tissue ◽

Lithium Chloride ◽

Osteoporosis Treatment ◽

Bone Morphogenetic Protein 2 ◽

Calcium Deposition ◽

Matrix Deposition ◽

Alp Activity

There is variability in the reported effects of compounds on osteoblasts arising from differences in experimental design and choice of cell type/origin. This makes it difficult to discern a compound’s action outside its original study and compare efficacy between compounds. Here, we investigated five compounds frequently reported as anabolic for osteoblasts (17β-estradiol (oestrogen), icariin, lactoferrin, lithium chloride, and menaquinone-4 (MK-4)) on human mesenchymal progenitors to assess their potential for bone tissue engineering with the aim of identifying a potential alternative to expensive recombinant growth factors such as bone morphogenetic protein 2 (BMP-2). Experiments were performed using the same culture conditions to allow direct comparison. The concentrations of compounds spanned two orders of magnitude to encompass the reported efficacious range and were applied continuously for 22 days. The effects on the proliferation (resazurin reduction and DNA quantification), osteogenic differentiation (alkaline phosphatase (ALP) activity), and mineralised matrix deposition (calcium and collagen quantification) were assessed. Of these compounds, only 10 µM MK-4 stimulated a significant anabolic response with 50% greater calcium deposition. Oestrogen and icariin had no significant effects, with the exception of 1 µM icariin, which increased the metabolic activity on days 8 and 22. 1000 µg/mL of lactoferrin and 10 mM lithium chloride both significantly reduced the mineralised matrix deposition in comparison to the vehicle control, despite the ALP activity being higher in lithium chloride-treated cells at day 15. This demonstrates that MK-4 is the most powerful stimulant of bone formation in hES-MPs of the compounds investigated, highlighting its potential in bone tissue engineering as a method of promoting bone formation, as well as its prospective use as an osteoporosis treatment.

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Effect of cell seeding density on matrix‐forming capacity of meniscus fibrochondrocytes and nasal chondrocytes in meniscus tissue engineering

The FASEB Journal ◽

10.1096/fj.201902559r ◽

2020 ◽

Vol 34 (4) ◽

pp. 5538-5551 ◽

Cited By ~ 1

Author(s):

Matthew Anderson‐Baron ◽

Melanie Kunze ◽

Aillette Mulet‐Sierra ◽

Adetola B. Adesida

Keyword(s):

Tissue Engineering ◽

Seeding Density ◽

Cell Seeding ◽

Cell Seeding Density ◽

Meniscus Tissue

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Synthesis, characterization and osteogenesis of phosphorylated methacrylamide chitosan hydrogels

RSC Advances ◽

10.1039/c8ra05378b ◽

2018 ◽

Vol 8 (63) ◽

pp. 36331-36337 ◽

Cited By ~ 2

Author(s):

Huishang Yang ◽

Shenggui Chen ◽

Lei Liu ◽

Chen Lai ◽

Xuetao Shi

Keyword(s):

Tissue Engineering ◽

Bone Formation ◽

Bone Tissue Engineering ◽

Bone Tissue ◽

Natural Bone ◽

Chitosan Hydrogels

Phosphorylated biopolymers can induce mineralization, mimic the process of natural bone formation, and have the potential as scaffolds for bone tissue engineering.

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The Effect of Simulated Microgravity by Three-Dimensional Clinostat on Bone Tissue Engineering

Cell Transplantation ◽

10.3727/000000005783982477 ◽

2005 ◽

Vol 14 (10) ◽

pp. 829-835 ◽

Cited By ~ 29

Author(s):

Masataka Nishikawa ◽

Hajime Ohgushi ◽

Noriyuki Tamai ◽

Koichi Osuga ◽

Masaru Uemura ◽

...

Keyword(s):

Tissue Engineering ◽

Bone Formation ◽

Bone Tissue Engineering ◽

Bone Tissue ◽

Three Dimensional ◽

Calcium Hydroxyapatite ◽

Osteoblastic Differentiation ◽

Sem Analysis ◽

Control Group ◽

Implant Surface

Evidence suggests that mechanical stress, including gravity, is associated with osteoblast differentiation and function. To examine effects of microgravity on bone tissue engineering, we used a three-dimensional (3D) clinostat manufactured by Mitsubishi Heavy Industries (Kobe, Japan). A 3D clinostat is a device that generates multidirectional G force. By controlled rotation on two axes, it cancels the cumulative gravity vector at the center of the device. We cultured rat marrow mesenchymal cells (MMCs) in the pores of interconnected porous calcium hydroxyapatite (IP-CHA) for 2 weeks in the presence of dexamethasone using the 3D clinostat (clinostat group). MMCs cultured using the 3D clinostat exhibited a 40% decrease in alkaline phosphatase activity (a marker of osteoblastic differentiation), compared with control static cultures (control group). SEM analysis revealed that although there was no difference between the two groups in number or distribution of cells in the pores, the clinostat group exhibited less extensive extracellular matrix formation than the control group. Cultured IP-CHA/MMC composites were then implanted into subcutaneous sites of syngeneic rats and harvested 8 weeks after implantation. All implants showed bone formation inside the pores, as indicated by decalcified histological sections and microfocus computed tomography. However, the volume of newly formed bone was significantly lower for the clinostat group than for the control group, especially in the superficial pores close to the implant surface. These results indicate that new bone formation in culture was inhibited by use of the 3D clinostat, and that this inhibition was mainly due to suppression of osteoblastic differentiation of MMCs.

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