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| No. | Author and year | Type of study | Subject/sample | Induction factor | WJ-MSC isolation method | Results | Conclusion |
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| 1. | Fu et al. 2018 [91] | In vitro and in vivo | (i) Human umbilical cord-derived mesenchymal stem cells (UC-MSCs)
(ii) Sprague-Dawley’s osteoblast
(iii) Sprague-Dawley’s osteoclast | Differentiation medium
Osteogenic medium:
(1) High-glucose DMEM
(2) 10% serum
(3) 10−8 M dexamethasone
(4) 50 ng/mL L-ascorbic acid
(5) 10 mM β-glycerophosphate | (i) Enzymatic digestion
(ii) Part of UC: not mentioned | (1) MicroCT result showed that transplantation of UC-MSCs increased bone mass in the distal condyle of normal rat femur compared to other groups
(2) Goldner’s staining indicated that compact arrangement of collagen with less trabecular thickness in the group undergoes implantation with UC-MSCs. A trabeculae-like structure containing lamellae was detected, but there are no cell arrangements found in the area
(3) Osteocalcin (OC) staining showed increased osteocalcin level in OVX-receiving UC-MSCs
(4) Antihuman-specific nuclei antigen showed engrafted UC-MSCs had differentiated into osteoblasts
(5) RT PCR:
(i) Human osteocalcin and abundant osterix (OSX) was detected in ovx-receiving UC-MSCs
(6) In vitro coculture system showed more expression of alkaline phosphatase (ALP) if the osteoblast is cocultured with UC-MSCs | UC-MSCs able to be differentiated into osteoblast and are safe for transplantation in bone disease treatment. |
Osteoclast differentiation
(1) 10 or 100 ng RANKL |
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| 2. | Al Jofi et al. 2018 [92] | In vitro | (i) Wharton’s jelly mesenchymal stem cells (WJ-MSCs) | (i) 10 μmol/L metformin (antidiabetic drug) | (i) Commercial UC-MSCs
(ii) Part of UC: not mentioned | Metformin-treated UC-MSCs increased in mineralization stained through Alizarin Red Staining. But in OCT-1- (organic cation transporter-) siRNA-transfected cells, a significant decrease in calcium-rich nodule formation was observed. | OCT-expressing WJ-MSCs have the ability to be differentiated into osteoblasts when induced with metformin. |
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| 3. | Bharti et al. (2018) [44] | In vitro | (i) Wharton’s jelly mesenchymal stem cells (WJ-MSCs) | (i) Osteogenic medium
(1) ADMEM
(2) 0.1 mM dexamethasone
(3) 50 mM ascorbic acid
(4) 10 mM glycerol-2-phosphate
(ii) Adipogenic medium:
(1) ADMEM
(2) 500 mM isobutyl methylxanthine
(3) 1 mM dexamethasone
(4) Insulin
(5) 100 mM indomethacin
(6) 10 mM insulin
(iii) Chondrogenic medium using comer
(1) StemPro1Osteocyte/Chondrocyte Differentiation Basal Medium; StemPro1 Chondrogenesis supplement; Gibco).
(2) Hepatocyte
(3) ADMEM
(4) 2% FBS
(5) 10 ng/ml of oncostatin M
(6) 10 nmol/L dexamethasone
(7) 1% insulin transferrin-selenium | (i) Explant method
(ii) Part of UC: maternal, middle and fetal segments | (1) Bone nodules: formed by cells from all segments
(2) Chondrogenic potential: occurred in cells from all segments | WJ-MSCs are a good cell source for autologous/allogeneic stem cell source. |
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| 4. | Batsali et al. 2017 [83] | In vitro | (i) Bone marrow mesenchymal stem cell (BM-MSCs)
(ii) Wharton’s jelly mesenchymal stem cells (WJ-MSCs) | Differentiation medium:
Osteogenic medium:
(1) High-glucose DMEM
(2) 10% serum
(3) 10−7 M dexamethasone
(4) 25 μg/mL L-ascorbic acid
(5) 3 mM NaH2PO4 | (i) Explant method
(ii) Part of UC: maternal, middle, and fetal segments | (1) Osteogenic differentiation (Alizarin Red and von Kossa staining): WJ-MSCs showed similar staining potential as BM-MSC
(2) Real-time- (RT-) PCR:
(i) Osteocyte-related gene expression: higher expression of Runt-related transcription factor 2 (RUNX2), distal-less homeobox protein 5 (DLX5), osteocalcin (OCN), and alkaline phosphatase (ALP) by BM-MSCs
(3) Differential expression of WNT ligands; sFRP4 (secreted frizzled-related protein 4) and WISP1 (WNT1-inducible signalling pathway protein 1) were significantly reduced in WJ-MSCs
(4) WISP1 implicated in osteogenic differentiation; RUNX2, ALP, and OSC were significantly upregulated | The osteogenic differentiation potential of WJ-MSCs is regulated by WISP1 and sFRP4, respectively. |
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| 5. | Zajdel et al. 2017 [84] | In vitro | (i) Adipose tissue (AT-MSCs)
(ii) WJ-MSCs | Osteogenic medium (Lonza):
(1) Dexamethasone
(2) Ascorbic acid
(3) β-Glycerophosphate | (i) Commercial AT-MSCs and UC-MSCs
(ii) Part of UC: not mentioned | (1) Calcium deposition by Alizarin Red staining
(i) Calcium deposition was greater in AT-MSCs when compared to WJ-MSCs
(2) ALP activity
(i) ALP activity was higher in AT-MSCs when compared to WJ-MSCs
(3) Osteoprotogerin (OPG) secretion
(i) Both osteo-induced cell types showed high OPG secretion when compared to control
(4) Osteocalcin (OC) secretion
(i) OC secretion was higher in WJ-MSCs when compared to AT-MSCs | WJ-MSCs have the ability to differentiate into the osteogenic lineage. |
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| 6. | Mechiche Alami et al. 2017 [88] | In vitro | (i) WJ-MSCs | Calcium phosphate (CaP) substrate build-up (without osteogenic induction | (i) Commercial UC-MSCs
(ii) Part of UC: not mentioned | (1) Gene expression analysis
(i) Day 7: Runt-related transcription factor 2 (RUNX2) and secreted phosphoprotein 1 (SPP-1) were upregulated
(ii) Day 14: collagen type 1 Alpha 1 (COL1A1) and ALP were upregulated
(iii) Day 21: bone gamma-carboxyglutamic acid-containing protein (BGLP) and ALP were upregulated
(2) Nodule characterization
(i) Hematoxylin-eosin-saffron (HES) staining revealed continuous layers of cells at the surface of the nodule with randomly distributed cells embedded within fibrous tissues
(ii) Masson’s trichrome staining showed the presence of green-stained fibrous tissue composed of newly formed collagen | Excellent osteogenic potential of sprayed CaP and WJ-MSCs in bone tissue engineering |
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| 7. | Szepesi et al. 2016 [61] | In vitro | (i) WJ-MSCs
(ii) AT-MSCs
(iii) Periodontal ligament MSCs (PDL-MSCs) | Differentiation medium:
(i) StemPro Osteogenesis Differentiation kit
(ii) StemPro Chondrogenesis Differentiation kit
(iii) StemPro Adipogenesis Differentiation kit
(iv) Endothelial Cell Growth Medium | (i) Enzymatic digestion UC-MSCs
(ii) Part of UC: not mentioned | (1) Osteogenic differentiation: AT-MSCs and PDL-MSCs showed greater calcium deposition
(2) Osteogenic differentiation assessed via RT-PCR:
(i) Runt-related transcription factor 2 (RUNX2): all cell types showed high expression after induction
(ii) Alkaline phosphatase (ALP): AT-MSCs and PDL-MSCs showed increased expression, but ALP was significantly lower in WJ-MSCs
(iii) Calcium deposition: AT-MSCs and PDL-MSCs showed greater calcium deposition as compared to WJ-MSCs
(3) There was a significant correlation between CD90 expression and the levels of calcium deposition in different MSC isolates | WJ-MSCs have osteogenic potential and are good cell sources for bone regeneration. |
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| 8. | Lim et al. 2016 [79] | In vitro | Human WJ-MSCs
(i) Fetal segment
(ii) Maternal segment
(iii) Middle segment | (i) Osteogenic medium
(1) Alpha-MEM
(2) Dexamethasone
(3) Ascorbic acid
(4) β-Glycerol phosphate
(ii) Adipogenic medium:
(1) DMEM/F12
(2) 3-Isobutyl-3-methylxanthine
(3) Dexamethasone
(4) Insulin
(iii) Chondrogenic medium
(1) Alpha-MEM
(2) Transforming growth factor 3 (TGF-β3) | (i) Enzymatic digestion
(ii) Maternal, middle, and fetal segments | Bone nodules: formed by cells from all segments | WJ-MSCs are a good cell source for bone regeneration. |
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| 9. | Kargozar et al. 2018 [80] | In vitro and in vivo | (i) BM-MSCs
(ii) AT-MSCs
(iii) UC-MSCs | (i) Nanocomposite scaffolds (3D bioactive glass/gelatin scaffolds (BaG/Gel) consisting of SiO2-P2O5-CaO (64% SiO2, 5% P2O5, and 31% CaO). | (i) Enzymatic digestion
(ii) Part of UC: not mentioned | In vitro study
(1) Cell viability:
(i) Scaffold had no significant inhibitory effect on MSC proliferation over time
(ii) MSC proliferation gradually increased with incubation time | BM-MSCs, grown on BaG/Gel nanocomposite scaffolds, are possible sources for bone regeneration. |
In vivo study
(1) Histological observations:
(i) H&E staining: all MSC-seeded scaffolds successfully generated new bone and demonstrated an ongoing healing process at 4 and 12 weeks after transplantation. The UC-MSC-seeded scaffold showed significantly increased neovascularization compared to the others
(ii) IHC staining: increased expression of OCN and ALP in the BM-MSC-seeded scaffold. Vascular endothelial growth factor (VEGF) expressed in all the groups of treated cell/scaffold. Increased neovascularization with the UC-MSC-seeded scaffold
(iii) Histomorphometry: BM-MSC-seeded scaffolds showed more bone regeneration at 4 and 12 weeks |
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| 10. | Todeschi et al. 2015 [81] | In vivo | (i) UC-MSCs
(ii) BM-MSCs | (i) Ceramic scaffolds (Skelite; 4 × 4 × 4 mm cubes of 33% hydroxyapatite and 67% silicon-stabilized tricalcium phosphate, Si-TCP)
(ii) Platelet-rich plasma (PRP)
(iii) Conditioned medium (CM) | (i) Explant method
(ii) Part of UC: not mentioned | (1) Histological assessment revealed the formation an immature bone-like structures and compact fibrous tissue in UC-MSC-seeded constructs
(2) Polarized light examination revealed less organized collagen fibers in the UC-MSC-seeded scaffolds. The immature bone-like matrix in UC-MSC-seeded scaffolds was mostly filled with a loose connective tissue
(3) Histological evaluation in an orthotopic mouse model showed that none of the bone defects had completely closed. However, gold MTC staining indicated the presence of red blood cells in blood vessel-like structures which is significant in the UC-MSC-transplanted group
(4) Osteocytes were clearly detectable in the BMMSC-seeded scaffolds
(5) However, human ALU sequences were not detected in osteocytes within the newly formed bone in the UC-MSC implants and nonseeded implants | UC-MSCs promote bone regeneration. |
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| 11. | Karadas et al. 2014 [74] | In vitro | (i) WJ-MSCs
(ii) BM-MSCs
(iii) Menstrual blood mesenchymal stem cells (MBMSCs) | (i) Collagen scaffolds with in situ calcium phosphate (CaP)
(ii) Differentiation medium:
(1) High-glucose DMEM
(2) 10 nM dexamethasone
(3) 50 μg/mL ascorbic acid
(4) 10 mM β-glycerophosphate
(5) 10% FBS
(6) 100 units/mL penicillin
(7) 100 μg/mL streptomycin | (i) Explant method
(ii) Part of UC: not mentioned | (1) Cell proliferation assays:
(i) WJ-MSCs on tissue culture polystyrene (TCPS) and collagen without CaP treatment increased proliferation
(2) Cell attachment (fluorescence staining):
(i) Good attachment of both cell types to the scaffold
(ii) Confocal micrographs showed that the cells were able to penetrate into the pores
(3) Osteogenic differentiation:
(i) ALP assay:
(a) WJ-MSCs showed better differentiation on untreated and CaP-containing foams than in growth medium
(b) ALP levels were higher in WJ-MSCs grown on CaP-free foams than on TCPS
(c) ALP activity was significantly higher in cells grown on collagen with CaP crystals formed in situ for both WJ-MSCs and MBMSCs (days 14 and 21) | Collagen foam with the use of CaP crystals formed in situ enhances the osteogenic induction of WJ-MSCs. |
von Kossa staining:
(1) WJ-MSCs had higher ALP activity and denser mineral deposition compared to MBMSCs |
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| 12. | Ramesh et al. 2014 [93] | In vitro | (i) WJ-MSCs | (i) Hydrogel alginate microspheres
(ii) Osteogenic differentiation media:
(1) Basal medium
(2) 10 mM β-glycerophosphate
(3) 1 mM dexamethasone
(4) 5 mg/mL ascorbic acid | (i) Explant method
(ii) Part of UC: not mentioned | (1) Characterization of osteodifferentiated WJ-MSCs via:
(i) Bradford assay: calcium deposition increased in 2% alginate
(ii) Alizarin Red staining: excellent matrix mineralization in WJ-MSCs immobilized on 2% alginate
(iii) Immunocytochemical analysis (osteocalcin): significant expression of osteocalcin in WJ-MSC aggregates in 1.5% and 2% alginate at day 21
(2) Genotypic analysis of encapsulated WJ-MSCs showed that OCN and Runx2 were upregulated in 1.5% and 2% alginate | WJ-MSCs encapsulated in hydrogel alginate microspheres have osteogenic potential for stem cell-based tissue engineering. |
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| 13. | Baba et al. 2012 [16] | In vivo & in vitro | Human umbilical cord mesenchymal stem cells (hUC-MSCs) | Differentiation medium:
(i) NH OsteoDiff Medium
(ii) NH AdipoDiff Medium
(iii) rhBMP2
(iv) Scaffold | (i) Enzymatic digestion
(ii) Part of UC: not mentioned | (3) Osteogenic differentiation: strong calcium deposition
Adipogenic differentiation: lipid droplet production
(4) H&E staining: positive for bone tissue producing osteocalcin (OCN)
(5) RT-PCR: high expression of RUNX2, ALP, and OCN as compared to undifferentiated cells | UC-MSCs supplemented with growth factors and serum have osteogenic differentiation potential for bone regeneration. |
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| 14. | Penolazzi et al. 2012 [89] | In vitro | (i) WJ-MSCs | (i) Porcine urinary bladder matrix (pUBM) | (i) Enzymatic digestion
(ii) Part of UC: not mentioned | (1) Proliferation assays showed
(i) pUBM did not have an effect on cells in suspension conditions but affected cells cultured in adherent conditions
(ii) Viable cells were homogenously distributed over the entire scaffold
(2) TUNEL assays showed
(i) No apoptosis in hWJ-MSCs cultured in agarose-coated wells with increasing amounts of pUBM
(ii) The scaffold upregulated cyclin D1
(iii) Matrix metanoproteinase (MMPI3) was lower but this did not affect β-catenin
(3) Morphological characterization:
(i) Scanning electron microscope (SEM) analysis showed
(a) There was a significant interaction between the cells and the biomaterial
(b) WJ-MSCs completely were enveloped in pUBM particles to form smooth spheroids, displaying an ECM network covering the surface
(c) The cells and the biomaterial formed a dense structure
(ii) X-ray energy-dispersive spectroscopy (EDX) showed that spheroids in osteogenic medium contained high amounts of calcium and phosphorous (high degree of mineralization)
(iii) Transmission electron microscopy (TEM) showed the presence of focal contacts between cells and the pUBM scaffold
(4) RT-PCR:
(i) RUNX2 expression was not affected by pUBM, but increased upon osteoinduction
(ii) WJ-MSCs seeded on pUBM were able to produce Col IAI and OPN after 21 days in osteogenic medium
(iii) WJ-MSCs showed increased ALP activity and ability to deposit mineralized matrix
(iv) OPN expression was higher in cells grown on pUBM scaffolds in osteogenic medium than in osteogenic medium alone | The combination of WJ-MSCs and pUBM shows the promise of scaffolds for bone regeneration. |
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| 15. | Wang et al. 2011 [94] | In vitro | (i) UC-MSCs | (i) Poly-L-lactic acid (PLLA) scaffold
(ii) Osteogenic induction medium:
(1) Low-glucose DMEM
(2) 10% FBS
(3) 1% penicillin/streptomycin
(4) 100 nM dexamethasone,
(5) 10 mM sodium β-glycerophosphate,
(6) 50 μg/mL ascorbic acid 2-phosphate (AA2P)
(7) 10 nM 1α,25-dihydroxyvitamin D3
(i) Chondrogenic induction medium:
(1) High-glucose DMEM (DMEM-HG)
(2) 1% nonessential amino acids (NEAA)
(3) 1x sodium pyruvate
(4) 1x insulin-transferrin-selenium premix (ITS)
(5) 50 μg/mL (AA2P)
(6) 40 μg/mL L-proline
(7) 100 nM dexamethasone
(8) 10 ng/mL TGF-β1 | (i) Enzymatic digestion
(ii) Part of UC: not mentioned | (1) Biochemical assays were performed to assess
(i) DNA content: osteogenic parts of the C-cell-O composites had higher DNA contents than the O-O composites and the osteogenic parts of the C-O composites
(ii) Glycosaminoglycan (GAG) content: all osteogenic groups had similar GAG contents
(iii) Hydroxyproline (HYP) content: osteogenic groups had a significantly higher HYP content
(iv) Calcium content: osteogenic groups showed significantly increased calcium levels over time
(2) Histological analyses: positive Alizarin Red staining in the osteogenic group
(3) RT-PCR:
(i) Collagen type II (ColII): not expressed in the osteogenic group
(ii) Collagen type I (ColI): upregulated in all groups
(iii) RUNX2: increased in the osteogenic group
(iv) Aggrecan: increased significantly in chondrogenic group | WJ-MSCs are a suitable cell source for a sandwich approach strategy in osteochondral tissue engineering. |
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| 16. | Schneider et al. 2010 [85] | In vitro | Human mesenchymal stem cells (hMSC):
(i) UC-MSCs
(ii) BM-MSCs | (i) Scaffold: 3D collagen gel
(ii) Osteogenic induction medium:
(1) Low-glucose DMEM
(2) 10% FCS
(3) 100 nM dexamethasone
(4) 10 mM sodium β-glycerophosphate
(5) 0.05 mM/L-ascorbic acid 2-phosphate
(iii) Adipogenic induction medium:
(1) DMEM high glucose
(2) 1 μM dexamethasone
(3) 0.2 mM indomethacin
(4) 0.01 mg/mL insulin
(5) 0.5 mM 3-isobutyl-1-methylxanthine
(6) 10% FCS | (i) Enzymatic digestion
(ii) Part of UC: not mentioned | (1) Scaffold:
(i) 3D collagen gel underwent progressive contraction
(ii) After osteogenic differentiation, collagen gels were stronger and harder with BM-MSCs and UC-MSCs
(2) Characterization of BM-MSCs and UC-MSCs by
(i) Osteogenic differentiation: UC-MSCs showed increased extracellular matrix (ECM) deposition by Alizarin Red (AR) staining
(ii) Adipogenic differentiation: UC-MSCs showed a limited number of small lipid vacuoles stained by Oil Red O (ORO)
(iii) Immunofluorescence: positive expression of collagen IV and laminin in UC-MSCs after 21 days of osteogenic differentiation on collagen gels
(iv) Immunohistochemistry analyses: positive expression of osteopontin (OPN) and bisphosphonate [2-(2-pyridinyl) ethylidene-BP] (PEBP) in the collagen gel
(3) TEM analysis:
(i) Osteogenic differentiation: contraction of the collagenous matrix on the UC-MSC-seeded collagen surface
(ii) Adipogenic differentiation: UC-MSC-derived lipid vacuoles were small and stained with toluidine blue
(4) RT-PCR: UC-MSCs expressed collagen I, collagen III, collagen IV, and laminin
(5) Cell migration: migrating UC-MSCs appeared as spindle-shaped cells with elongated cytoplasmic processes | UC-MSCs have a significant therapeutic impact in bone tissue engineering in the future. |
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| 17. | Hsieh et al. 2010 [14] | (i) In vitro | (i) WJ-MSCs
(ii) BM-MSCs | Osteogenic differentiation medium:
(1) DMEM
(2) 10% FBS
(3) 0.1 mM dexamethasone
(4) 10 mM β-glycerophosphate
(5) 50 mM ascorbic acid | (i) Enzymatic digestion
(ii) Part of UC: not mentioned | (1) Array data showed that both BM-MSCs and WJ-MSCs expressed multilineage differentiation properties.
(2) Real-time-PCR:
BM-MSCs > WJ-MSCs in terms of
(i) Adipocytic marker expression: lipoprotein lipase (LPL), leptin, peroxisome proliferator-activated receptor gamma (PPARγ), and fatty acid-binding protein 4 (FABP4)
(ii) Lipid accumulation
(3) Osteogenic differentiation potential
(i) BM-MSCs > WJ-MSCs when cultured in defined MesenCult
(ii) Expressed higher levels of ALP, SPP-1, and RUNX2
(iii) BM-MSCs > WJ-MSCs when cultured in defined cultured in 10% FBS:
(a) Expressed higher levels of ALP, osteopontin, and RUNX2
(4) ALP staining (better osteogenic ability) | WJ-MSCs are capable of differentiating into the osteogenic lineage, but BM-MSCs are superior. |
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| 18. | Hou et al. 2009 [15] | (i) In vitro | (i) hUC-MSCs
(ii) BM-MSCs | (i) BMP-2 treatment:
(a) Bone morphogenetic protein 2-blocking antibodies (BMP2 Ab)
(b) Recombinant human BMP2 (rhBMP2)
(c) Noggin
(ii) Osteogenic differentiation medium:
(1) DMEM/F12
(2) 10% FBS
(3) Dexamethasone
(4) Ascorbic acid (AsA) 2-phosphate
(5) β-Glycerophosphate
(iii) Adipogenic differentiation medium:
(1) DMEM/F12
(2) 1% FBS
(3) 100 nM dexamethasone
(4) 1 nM insulin
(iv) Chondrogenic differentiation medium:
(1) DMEM/F12
(2) Dexamethasone
(3) AsA
(4) TGF-β1
(5) ITS+ Premix | (i) Enzymatic digestion
(ii) Part of UC: not mentioned | (1) Trilineage differentiation
(i) Osteogenesis: osteonectin, ALP, and RUNX2 were expressed
(ii) Chondrogenesis: COL II, collagen type X (COL X), and aggrecan were expressed
(iii) Adipogenesis: adipsin, PPARγ, and lipoprotein lipase
(2) ALP activity was significantly increased in BMP2-induced UC-MSCs
(i) Western blot revealed activation of the BMP2 signaling pathway in both cell types via SMADs, p38, and extracellular regulated kinase activation | BMP2-induced UC-MSCs have good osteogenic differentiation (indicated by the activation of BMP2 signaling) and may be used in tissue-engineered bone. |
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