Osteogenic Induction of Wharton s Jelly-Derived ...downloads.hindawi.com/journals/sci/2018/2406462.pdf · Review Article Osteogenic Induction of Wharton’s Jelly-Derived Mesenchymal

Review ArticleOsteogenic Induction of Wharton’s Jelly-Derived MesenchymalStem Cell for Bone Regeneration: A Systematic Review

Ayu Suraya Ansari,1 Muhammad Dain Yazid,2 Nur Qisya Afifah Veronica Sainik,1

Rabiatul Adawiyah Razali,1 Aminuddin Bin Saim,3 and Ruszymah Bt Hj Idrus 1

1Department of Physiology, Faculty of Medicine, Universiti Kebangsaan Malaysia Medical Centre, Jalan Yaacob Latif, 56000 Cheras,Kuala Lumpur, Malaysia2Tissue Engineering Centre, Universiti Kebangsaan Malaysia Medical Centre, Jalan Yaacob Latif, 56000 Cheras,Kuala Lumpur, Malaysia3Ear, Nose & Throat Consultant Clinic, Ampang Puteri Specialist Hospital, 68000 Ampang, Selangor, Malaysia

Correspondence should be addressed to Ruszymah Bt Hj Idrus; [email protected]

Received 19 May 2018; Revised 27 July 2018; Accepted 3 September 2018; Published 11 November 2018

Academic Editor: Jeong-Chae Lee

Copyright © 2018 Ayu Suraya Ansari et al. This is an open access article distributed under the Creative Commons AttributionLicense, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work isproperly cited.

Wharton’s jelly-derived mesenchymal stem cells (WJ-MSCs) are emerging as a promising source for bone regeneration in thetreatment of bone defects. Previous studies have reported the ability of WJ-MSCs to be induced into the osteogenic lineage. Thepurpose of this review was to systematically assess the potential of WJ-MSC differentiation into the osteogenic lineage. Acomprehensive search was conducted in Medline via Ebscohost and Scopus, where relevant studies published between 1961 and2018 were selected. The main inclusion criteria were that articles must be primary studies published in English evaluatingosteogenic induction of WJ-MSCs. The literature search identified 92 related articles, but only 18 articles met the inclusioncriteria. These include two animal studies, three articles containing both in vitro and in vivo assessments, and 13 articles onin vitro studies, all of which are discussed in this review. There were two types of osteogenic induction used in these studies,either chemical or physical. The studies demonstrate that WJ-MSCs are able to differentiate into osteogenic lineage and promoteosteogenesis. In light of these observations, it is suggested that WJ-MSCs can be a potential source of stem cells for osteogenicinduction, as an alternative to bone marrow-derived mesenchymal stem cells.

1. Introduction

1.1. Human Umbilical Cord. About 25 years ago, the umbili-cal cord was considered to be a type of medical waste,until it was found to be a rich source of stem cells [1].The abundance of stem cells and the ease of isolation havebecome deciding factors while choosing the source of adultstem cells. The human umbilical cord (HUC) is approxi-mately 65 cm in length and 1.5 cm in diameter [2, 3]; itconnects the fetus to the mother and supplies nourish-ment. The cord is covered by single or multiple layer ofsquamous cubic epithelial cells derived from the develop-ing amnion [4–6]. The HUC contains two arteries andone vein, which are surrounded by a mucoid connectivetissue known as Wharton’s jelly (WJ) [7]. This mucous

connective tissue is made up of mucopolysaccharides thatare hyaluronic acid and chondroitin sulphate [8]. Basi-cally, WJ can be divided into 4 layers with the outerlayer being the amniotic epithelium followed by cord lin-ing WJ and intermediate WJ. The inner layer of WJ isalso known as perivascular WJ which contains arteriesand vein [7] (Figure 1).

It has been reported that WJ also contains myofibroblast-like stromal cells, collagen fibers, proteoglycans, fibroblasts,and macrophages. WJ has garnered interest due to its avail-ability, the noninvasive method of collection, and high cellyields. It has been demonstrated that mesenchymal stem cells(MSCs) isolated from the umbilical cord express matrixreceptors (CD44 and CD105) and integrins (CD29 andCD51), but not hematopoietic lineage markers (CD34 and

HindawiStem Cells InternationalVolume 2018, Article ID 2406462, 17 pageshttps://doi.org/10.1155/2018/2406462

http://orcid.org/0000-0003-1060-4273

https://creativecommons.org/licenses/by/4.0/

https://creativecommons.org/licenses/by/4.0/

https://doi.org/10.1155/2018/2406462

CD45) [9]. These cells exhibit a phenotype similar to that ofmesenchymal stem cells from other tissue sources [10].According to the International Society for Cellular Therapy(ISCT), stem cells should demonstrate plastic adherence,not expressing hematopoietic markers, and be able to com-mit to the adipogenic, osteogenic, and chondrogenic lineage[11]. WJ-MSCs have a huge advantage where the phenotypeand stemness are remained despite being in a long-term cul-ture. This enables a mass production of cells which is usuallyrequired for regenerative medicine [12].

1.2. WJ-MSCs and Their Potential in the Treatment ofDiseases. WJ, also known as substantia gelatinea funiculiumbilicalis, consists of fibroblast-like cells and mast cells thatare embedded in proteoglycans, mainly hyaluronic acid. Thecells are thought to be trapped in WJ during the early stage ofembryogenesis, when they migrate from the aortic gonado-tropin mesonephric region to the fetal liver through theumbilical cord [13]. WJ-MSCs have many advantages overother types of stem cells, including higher proliferation ratesand broader multipotency. WJ-MSCs are able to differentiateinto many cell types such as adipocytes [14, 15], osteoblasts[14, 16], hepatocytes [17], chondrocytes [18], and neural cells[19, 20]. Interestingly, it has also been demonstrated in athree-dimensional model that WJ-MSCs are able to differen-tiate in vitro into cornea epithelial-like cells, which may offera solution for patients with limbus stem cell deficiency [21].

Cui and colleagues have successfully improved cogni-tive function in a mouse model of Alzheimer’s diseaseusing intravenously delivered WJ-MSCs, which reducedoxidative stress and promoted hippocampal neurogenesis[20]. In a clinical trial conducted by Hu and colleagues,an intravenous infusion of WJ-MSCs in type 2 diabetesmellitus patients improved the function of islet β-cellsand reduced the incidence of diabetic complications [22].Additionally, WJ-MSC therapy is now used to treat cor-neal epithelial, stromal, and endothelial disorders apart

from the conventional intervention such as surgery, ioniz-ing radiation, or drug treatment [23, 24].

1.3. Immunomodulatory Aspect of WJ-MSCs and BM-MSCs.WJ harbours MSCs that possess a similar phenotype asharvested from the bone marrow and other sources. WJ-MSCs do not express HLA-DR and costimulatory moleculesCD40, CD80, and CD86 which are essential for the activationof T-cells [1, 2, 9, 25–27]. WJ-MSCs have been proven tohave lower immunogenicity than BM-MSCs as depicted byWeiss et al. [1] who conducted an experiment using mixedlymphocyte reaction (MCR) assay [26]. In a study conductedby Prasanna et al. and Deuse et al., both consistently showedlow HLA-DR expression compared to BM-MSCs after stim-ulation with IFN-γ and proinflammatory cytokines [28, 29].In normal culture conditions, HLA-DR is not expressed;hence, the activation of T-cell is inhibited, reducing the riskof allograft rejection which potentially makes it safe forhuman transplantation.

It is documented that BM-MSCs harbour viruses, whichis a major drawback in clinical application. There are reportsfrom patients who undergo BM-MSC transplant who areinfected with viral infection as a complication of the cell-based therapy [9]. Interestingly, the virus can escape fromdetection therefore increasing the morbidity and mortality[30]. Moreover, there are various diseases such as aplasticanemia, leukaemia, and bone marrow failure that impedesthe application of BM-MSCs in therapy [31].

1.4. WJ-MSC Homing and Migration for Bone Healing. Gen-erally, MSCs are known to migrate towards the injury siteand help the healing process. The migration process,known as homing, is defined as the arrest of MSCs withinthe vasculature of a tissue before it crosses over the endo-thelium [24, 32]. As for now, the mechanism of MSChoming is still vague and BM-MSCs are postulated to havea mechanism similar to leukocyte homing. The mechanism

INNER LAYER

OUTER LAYER

Amniotic epithelium

Cord lining Wharton’s jelly

Intermediate Wharton’s jelly

Perivascular Wharton’s jelly

Umbilical vein

Umbilical arteries

Umbilicalarteries

Umbilicalvein

Wharton’sjelly

Figure 1: Anatomical compartment of Wharton’s jelly mesenchymal stem cell.

2 Stem Cells International

is initiated when MSCs collide with the endothelium roll-ing, causing a slackening of cells in the blood flow. The G-protein-coupled receptors activated the cells and activatedintegrin mediation, causing activation-dependent arrest.The process is completed with the transmigration of thecells through the endothelium and the underlying base-ment membrane [33]. There are several factors thatcontribute to the homing mechanism which are growthfactor expression as well as chemokine and extracellularmatrix receptors on the MSCs’ surfaces [34].

In a study conducted by Granero-Moltó et al., MSCs aremigrated to the fracture site via the CXCR4 receptor causingimprovement of biomechanical properties and increasing thecartilage and bone of the callus [24]. Zwingenberger et al.demonstrated that the combination of the SDF-1 releasedand bone morphogenetic protein 2 contributes to the migra-tion of the stem cell [35]. This occurrence markedly boostsbone regeneration. Apart from that, MSCs are also recruitedtowards wear-particle-related osteolysis which is indicated bythe inflammatory macrophage that is also the chemokine CCreceptor (CCR) 1 of MSCs [30, 36]. The MSCs are demon-strated to increase bone mineral density and decrease theosteolytic process [37].

1.5. Cellular Mechanism of Bone Remodelling for BoneRegeneration. Bone repair or bone regeneration is character-ized by a series of tissue transformation mechanisms includ-ing resorption and formation of hard and soft tissue.Therefore, mineralized tissue remodelling is required forthe involvement of various cell types including osteoclastand osteoblast. Osteoblasts are bone-forming cells that canbe found at the surface of bone, while osteoclasts are multi-nucleated bone-resorbing cells derived from bone marrowstem cells [38]. Bone remodelling is a cyclical process inwhich bone undergoes consistent renewal to ensure thereplacement of primary bone, to maintain calcium homeo-stasis, and especially to heal ischemic and microfracturedbone [7, 20, 32–34, 39]. This process requires a correct bal-ance of bone resorption and bone formation and thusinvolves osteoclasts and osteoblasts, respectively.

Bone remodelling consists of five consecutive phases: (1)the resorption phase, where osteoclasts break down the bonetissue, resulting in mineral release; (2) the reversal phase,where mononuclear cells appear on the bone surface; (3)the formation phase, where osteoblasts trapped in the bonematrix become osteocytes; (4) the mineralization phase,where osteocytes produce type I collagen and other sub-stances that make up the bone extracellular matrix; and (5)the termination phase [38, 40]. Resorption is initiated byosteoclast progenitors that are recruited and disseminatedinto the bloodstream. These cells proliferate and differentiateinto mature osteoclasts, aided by osteoblast stromal cells viacell-to-cell interactions. These osteoblasts express two cyto-kines, i.e., receptor activator of NF-κB ligand (RANKL) andosteoprotegrin (OPG), involved in osteoclast progenitor celldifferentiation. Under parathyroid hormone (PTH) stimula-tion, RANKL will bind to RANK, a cytoplasmic membranereceptor on osteoclast progenitor cells, to stimulate theirfusion, differentiation, and activation. In contrast, OPG

binds to RANKL to counterbalance the effect of RANKL-RANK, which thereby determines the extent of bone resorp-tion. These events are important in maintaining bonehomeostasis [41] (Figure 2). Bone resorption is terminatedwhen osteoclasts undergo apoptosis and the reversal phaseis initiated. Reversal cells may thus represent the missing linknecessary to understand the coupling between bone resorp-tion and formation. Researchers have found that reversalcells colonizing the resorbed bone surface are immature oste-oblastic cells that gradually mature into bone-forming osteo-blasts during the reversal phase and prepare the bone surfacefor bone formation [42].

1.6. Molecular Mechanism of Bone Remodelling for BoneRegeneration. The differentiation of MSCs depends on whichsignalling pathway is activated. Apart from osteoblasts, WJ-MSCs have also been demonstrated to differentiate into othermesenchymal cell lineages such as hepatocytes [43], chon-drocytes [18, 44], and adipocytes [14, 15]. The markers forosteogenic differentiation are alkaline phosphatase (ALP),an early marker of osteogenic differentiation and mineraliza-tion, and RUNX2, a runt domain-containing transcriptionfactor that is crucial for osteogenic differentiation and boneformation. The activation of RUNX2 triggers COL1 (collagentype 1), osteopontin (OPN), and osteocalcin (OC), which areosteoblast-specific markers. OPN is expressed later in the dif-ferentiation stage [45, 46]. Both OPN and COL1 are synthe-sized by osteoblasts. OC is also expressed later and isimportant for maintaining bone resorption. Osterix (Osx) isa downstream factor of Runx2 that binds to activated NFAT2in bone development [47]. A study by Zhou et al. exploredthe function of Osx where it regulates bone homeostasis afterbirth for bone and cartilage formation [26].

In osteoblasts, lineage-specific gene expression controlby specific transcription factors, i.e., Cbfa-1/RUNX2, actsto regulate osteoblastic specific gene expression [48]. Cbfa-1/RUNX2 is required for osteoblast differentiation, sinceCbfa-1 knockout mice display impaired or even absent boneformation [49, 50]. This transcription factor contains a runtDNA-binding domain, which can bind to DNA as a mono-mer or as a subunit of a monomeric complex. It binds to var-ious enhancers and promoters, including those for the genesencoding osteocalcin, osteopontin, bone sialoprotein, andGM-CSF. The expression of these proteins contributes tothe bone matrix, leading to the maturation of osteoblasts.These genes can also be used as markers for different stagesof osteoblast development [51–53].

The expression of transcription factors is controlled byseveral pathways that are activated by growth factors (GFs)that bind to a specific receptor. These growth factors includefibroblast growth factor (FGF), transforming growth factor-β(TGF-β), insulin-like growth factor (IGF), platelet-derivedgrowth factor (PDGF), and vascular endothelial growth fac-tor (VEGF) [54]. It has been reported that these GFs areresponsible for regulating the expression of Cbfa-1/RUNX2via the MAPK [55], ERK [52], and PI3K-Akt pathways [54,56]. GF binding to its receptor tyrosine kinase (RTK) acti-vates a downstream signaling cascade. The activated RTKactivates class I phosphatidylinositol 3-kinase (PI3K) or

3Stem Cells International

guanosine nucleotide-binding protein (Ras) and propagatesthe signal through direct binding or tyrosine phosphoryla-tion. This then activates Akt/PKB, IκK/IκB, or Raf/MEK,which then activates NF-κB or MAPK, accordingly. Acti-vated NF-κB and MAPK act through direct binding to phos-phorylate ERK/JNK-cJun, which then activate Cbfa1/RUNX2 gene expression. TGF-β plays important roles inosteoblast precursor recruitment, FGF enhances osteoblastrecruitment and proliferation, IGF is involved in the regula-tion of bone matrix synthesis and migration, VEGF regulatesosteoblast differentiation, and PDGF is involved in osteopro-genitor migration [51, 52]. Cumulatively, the osteogenic dif-ferentiation capability owned by WJ-MSCs supplementedwith specific GF is postulated to have a high potential forbone regeneration.

1.7. WJ-MSCs for Bone Regeneration. Albeit many have usedWJ-MSCs in the studies, the safety and efficacy of its applica-tion are indecisive particularly in bone regeneration. Thereare several aspects that need to be considered prior to usingWJ-MSCs that may influence the yield and its stemnesspotency. Different parts of the umbilical cord generateddiverse frequencies of MSCs and cell populations [6–8, 57–59]. It is known that Runx2 plays a pivotal role in osteoblastdifferentiation. In a previous study, it has shown that WJ-MSCs have lower capability to differentiate due to the highlevel of RUNX2 but lower ALP expression. ALP is importantfor matrix maturation [60, 61]. WJ-MSCs also exhibited

higher expression of pluripotent markers, OCT 4, SOX 2,and NANOG than in other parts of the umbilical cord [62].From those findings, it can be postulated that regulation ofRunx2 and pluripotent impedes ALP expression, thus need-ing a specific modulator that can serve as a molecular switchof WJ-MSCs’ fate. Current study by Bustos and colleagueshave demonstrated that JARID1B (Jumonji AT-rich interac-tive domain 1B) histone demethylase represses Runx2 inundifferentiatedWJ-MSCs. In JARID1B knockdown murine,it can be seen that Runx2 is highly expressed and ready forosteogenic commitment indicating that this molecularmechanism is relevant to modulating osteoblastic lineagecommitment [63].

1.8. Clinical Application of WJ-MSCs in Bone Regeneration.The current standard commonly used for bone tissuereplacement is bone grafting obtained from patientsthemselves (autograft) or from other individuals (allograft).However, this has raised various effects including immunore-activity and infection as well as procedure. WJ-MSCs haveproved its capability to help in bone regeneration for clinicalapplication. Qu et al. treated 36 patients with nonunion bonefracture with WJ-MSCs cultured with platelet-rich plasma(PRP) resulting in a faster recovery with no infectionrecorded compared to the other 36 patients with autoiliactreatment [64]. In another study, the intravenous injectionof 3–5 million WJ-MSCs alleviated the condition of Beckermuscular dystrophy patients with increased muscle strength,

Cbfa-1

OCNOPNBSPGM-CSF

Cbfa-1c-Jun

Cbfa-1NF-𝜅B

Akt

Ras/MEF

MAPK

ERK/JNK-c-Jun

Osteoblast

Area of bone regeneration

FGF TGF-𝛽IGF PDGF

PI3K

I𝜅K/I𝜅B

NF-𝜅B

Osteoclastprogenitor Osteoclast

RANKRANKLOPG

FusionNo fusion

Figure 2: Mechanism of bone regeneration and activation of signaling pathways.


improved appetite, and also improved patient’s gait [65].Recently, a patient in Indonesia with infected nonunion boneis able to walk with no pain and no postoperative compli-cations recorded after local implantation of 5 million cellssupplemented with BMP-2 and hydroxyapatite [66]. Atransplantation of autologous BM-MSCs and allogeneicWJ-MSCs in treating osteonecrosis of femoral headshowed improvement where it relieved the pain andimproved the joint function [67].

To this date, there are many various positive outcomesupon WJ-MSC treatment in clinical trials for different dis-orders including neurology [67], hematology [68], liverdiseases [69], and particularly musculoskeletal diseases[65, 70]. WJ-MSCs have a huge potential to be used asan alternative treatment for bone disorders as it does notrequire ethical issues to obtain and it is alleviatingpatients’ morbidities [71]. Since WJ-MSCs can retain thestemness and phenotypic stability compared to BM-MSCs,it has potency to be commercialised [72].

2. Methods

2.1. Search Strategy. A systematic review was conducted tosystematically assess articles on the potential of WJ-MSCsfor bone regeneration. Two databases were comprehensivelyused to search for relevant studies, i.e., Medline via Ebscohostand Scopus. For search term keywords, the combination ofwords used was “Wharton’s jelly” AND “osteo∗” OR “bone”.

2.2. Selection Criteria. The year limit for searches was from1961 to 2018, and only studies published in English wereconsidered. The search outcomes identified all articlescontaining the words Wharton’s jelly, umbilical cord, osteo-genesis, osteogenic, and bone. Databases were searched indi-vidually to ensure all relevant studies were considered. Thetitles and abstracts were carefully screened for eligibilityrelated to the topic of interest. Primary studies related tobone formation or bone regeneration were included. Reviewarticles, news articles, letters, editorials, and case studies wereexcluded from the search.

2.3. Data Extraction and Management. Data were extractedfrom each eligible article by two reviewers. The selectedpapers were screened in several phases prior to inclusion.First, the titles that were not relevant to the topic wereexcluded. Next, the abstracts of the papers were screened,and unrelated studies were excluded. All duplicates wereremoved. The following data were summarized from theselected studies: (1) authors, (2) type of study, (3) subject/sample, (4) induction factor, (5) methodology, (6) results,and (7) conclusions.

3. Results

3.1. Search Results. The primary searches identified 386 arti-cles: 41 articles came from Medline and 345 articles werefound in Scopus. To minimize bias and improve the strengthof the related articles, two reviewers independently assessedthe articles according to the inclusion and exclusion criteria.There were 244 articles removed as they were unrelated to

either Wharton’s jelly or osteogenesis/bone. A joint discus-sion was conducted to achieve consensus where differencesemerged during the assessment. From the 142 remainingarticles, 50 duplicates were removed before full articles wereretrieved. From 92 articles, 74 articles were rejected basedon the inclusion criteria as the articles were not primary stud-ies, were not related to Wharton’s jelly or osteogenesis, orwere not available as full articles. Finally, a total of 18 studieswere selected for data extraction in this review. The flowchart of the selection process is shown in Figure 3.

3.2. Study Characteristics. All studies were published between1961 and 2018. An article reported on animal studies(in vivo), two articles on both in vitro and in vivo assess-ments, and 14 articles on in vitro studies. There were six arti-cles which included scaffold fabrication in the study. Threeout of six scaffold studies involved the optimization of thescaffold component for better bone regeneration. Elevenstudies proposed a different type of tissue as the MSC source.From the generated data, we classified the articles into twosubgroups: (1) chemical methods to promote osteogenesisand (2) physical methods (scaffolds) to promote osteogene-sis. A summary of the studies is provided in Table 1.

4. Discussion

The database search provided 18 articles related to Whar-ton’s jelly, umbilical cord, osteogenesis, osteogenic lineage,and bone. From these articles, various tissue sources wereassessed for potential MSCs. Each of these sources was exam-ined regarding MSC differentiation capacities into the adipo-genic, chondrogenic, and osteogenic lineages. This reviewassessed the osteogenic potential of WJ-MSCs, which mayhave remarkable potential for bone regeneration in the clinic.

4.1. Mesenchymal Stem Cells: Potential Sources. Mesenchy-mal stem cells (MSCs) have attracted attention because oftheir unique plasticity and ability to differentiate into multi-ple cell lineages, i.e., osteoblasts, chondrocytes, and adipo-cytes, with potential for clinical usage. The bone marrow isa primary source of MSCs. However, it has been reportedthat the frequency as well as the differentiation potential ofBM-derived MSCs (BM-MSCs) decline with increasing age[73]. Therefore, alternative sources of MSCs are needed,especially those that can be obtained noninvasively. Cur-rently, various tissues are under consideration for MSC isola-tion, including adipose tissue, muscle, amniotic fluid,menstrual blood [74, 75], fetal blood [76], and periodontalligaments (PDL) [61]. The human umbilical cord is a prom-ising source of MSCs, as MSCs can be isolated either from thewhole umbilical cord [16, 77], the umbilical vein subendothe-lium [78], or WJ [14, 19, 74]. One group of researchersdivided the umbilical cord into three anatomical segments,i.e., the maternal, middle, and fetal segments [79]. They dem-onstrated that MSCs from the maternal and fetal segmentsdisplayed greater viability, possessed significantly higher pro-liferation rates, and underwent more complete osteogenicdifferentiation, showing that these segments are a goodsource of MSCs for bone tissue engineering [79].


It is important to characterize cells derived from tissuesto determine the type of cell population that exists in thepreparation. A heterogeneous population could influencethe differentiation properties, specifically the osteogenicpotential of MSCs for bone regeneration. There are a few sur-face markers that are commonly reported for MSCs such asCD13, CD29, CD44, CD73, CD90, CD105, and CD166.MSCs do not express CD31, CD144, and CD309 (endothelialcell markers) or CD14, CD34, CD45, CD117, and CD133(hematopoietic cell markers) [61, 79].

4.2. Wharton’s Jelly Mesenchymal Stem Cells UndergoOsteogenic Differentiation. WJ-MSCs have been shown tohave good potential for osteogenic differentiation. These cellsdisplay all features of functional osteocytes/osteoblasts basedon osteogenic gene expression, extracellular matrix (ECM)mineralization, and the ability to adhere to a fabricatedscaffold [80, 81]. Although WJ-MSCs have been broadlyinvestigated, there are still problems when it comes to trans-plantation, as an immune response and rejection could occur[82]. In the database search, we only found one article thatused autoserum for WJ-MSCs in vitro as a substitute forFBS to reduce the rejection rate. Autoserum is serumobtained from the umbilical cord blood. Baba et al. reported

that the cell culture medium using autologous serum is supe-rior in quality to medium using FBS. WJ-MSCs cultured inautologous serum exhibited successful osteoblastic andadipogenic differentiation. The WJ-MSCs were then trans-planted subcutaneously into nude mice, and their potentialto form bone was proven [16].

4.2.1. Chemical Induction. Seven studies out of 18 selectedarticles used chemical factors to promote osteogenesis inWJ-MSCs. Batsali et al. [83] demonstrated that WJ-MSCsare able to differentiate into the osteogenic lineage,although this was inferior compared to BM-MSCs. Theydemonstrated that WISP1, a canonical Wnt pathway targetprotein, was able to promote better osteogenic differentia-tion in WJ-MSCs [83]. A study by Szepesi et al. [61]showed that adipose tissue-derived mesenchymal stemcells (AT-MSCs) and periodontal ligament-derived mesen-chymal stem cells (PDL-MSCs) have excellent potential forbone replacement applications and better endothelial dif-ferentiation ability as compared to WJ-MSCs [61]. Thehigh degree of calcification in AT-MSCs and PDL-MSCsdemonstrates that calcium deposition was better as com-pared to WJ-MSCs for generating vascularized bone grafts[61]. Similar findings were reported by [84] where AD-

Search of electronic databasesMedline = 41, Scopus = 345

Total = 386

Identification of abstractsMedline = 94, Scopus = 48

Total = 142

Removal of abstracts due to duplicates = 50

Primary screening of abstracts = 92

Rejection of abstracts based on selection criteria(74 articles)

(i) Not primary studies = 53(ii) Not related with Wharton’s jelly and/or

osteogenesis = 14(iii) Not a full article = 7

Selected abstractsScopus = 6

Medline =12

Full articles obtainedScopus = 6

Medline = 12Total = 18

Exclusion of unrelated papers = 244

Figure 3: Flow chart of the article selection process using the Scopus and MEDLINE databases.


Table1:Summaryandclassification

ofthe18

articles

selected

from

thedatabase

search.

No.

Autho

rand

year

Typeof

stud

ySubject/sample

Indu

ctionfactor

WJ-MSC

isolation

metho

dResults

Con

clusion

1.Fu

etal.

2018

[91]

Invitro

and

invivo

(i)Hum

anum

bilical

cord-derived

mesenchym

alstem

cells

(UC-M

SCs)

(ii)Sprague-Daw

ley’s

osteoblast

(iii)

Sprague-Daw

ley’s

osteoclast

Differentiationmedium

Osteogenicmedium:

(1)High-glucoseDMEM

(2)10%

serum

(3)10

−8M

dexamethasone

(4)50

ng/m

LL-ascorbicacid

(5)10

mM

β-glyceroph

osph

ate

(i)Enzym

atic

digestion

(ii)Partof

UC:n

otmention

ed

(1)MicroCTresultshow

edthat

transplantationof

UC-M

SCsincreased

bone

massin

thedistalcond

yleof

norm

alratfemur

comparedto

other

grou

ps(2)Goldn

er’sstaining

indicatedthatcompactarrangem

entof

collagenwith

lesstrabecular

thickn

essin

thegrou

pun

dergoesim

plantation

withUC-

MSC

s.Atrabeculae-likestructurecontaining

lamellaewas

detected,but

thereareno

cellarrangem

entsfoun

din

thearea

(3)Osteocalcin

(OC)staining

show

edincreasedosteocalcinlevelinOVX-

receivingUC-M

SCs

(4)Antihum

an-specificnu

cleiantigenshow

edengraftedUC-M

SCshad

differentiated

into

osteoblasts

(5)RTPCR:

(i)Hum

anosteocalcinandabun

dant

osterix(O

SX)was

detected

inovx-

receivingUC-M

SCs

(6)In

vitrococultu

resystem

show

edmoreexpression

ofalkalin

eph

osph

atase(A

LP)iftheosteoblastiscocultured

withUC-M

SCs

UC-M

SCsableto

bedifferentiated

into

osteoblast

andaresafefor

transplantationin

bone

diseasetreatm

ent.

Osteoclastdifferentiation

(1)10

or100ng

RANKL

2.AlJofi

etal.

2018

[92]

Invitro

(i)Wharton

’sjelly

mesenchym

alstem

cells

(WJ-MSC

s)

(i)10

μmol/L

metform

in(antidiabetic

drug)

(i)Com

mercialUC-

MSC

s(ii)Partof

UC:n

otmention

ed

Metform

in-treated

UC-M

SCsincreasedin

mineralizationstainedthrough

Alizarin

Red

Staining.B

utin

OCT-1-(organiccation

transporter-)siRNA-

transfectedcells,a

significant

decrease

incalcium-richno

duleform

ation

was

observed.

OCT-expressingWJ-MSC

shave

theability

tobe

differentiated

into

osteoblastswhenindu

ced

withmetform

in.

3.Bhartietal.

(2018)

[44]

Invitro

(i)Wharton

’sjelly

mesenchym

alstem

cells

(WJ-MSC

s)

(i)Osteogenicmedium

(1)ADMEM

(2)0.1mM

dexamethasone

(3)50

mM

ascorbicacid

(4)10

mM

glycerol-2-pho

sphate

(ii)Adipo

genicmedium:

(1)ADMEM

(2)500mM

isobutyl

methylxanthine

(3)1mM

dexamethasone

(4)Insulin

(5)100mM

indo

methacin

(6)10

mM

insulin

(iii)

Cho

ndrogenicmedium

using

comer

(1)Stem

Pro1O

steocyte/

Cho

ndrocyte

DifferentiationBasal

Medium;StemPro1

Cho

ndrogenesissupp

lement;

Gibco).

(2)Hepatocyte

(3)ADMEM

(4)2%

FBS

(5)10

ng/m

lofon

costatin

M(6)10

nmol/L

dexamethasone

(7)1%

insulin

transferrin-selenium

(i)Explant

metho

d(ii)Partof

UC:

maternal,middle

andfetal

segm

ents

(1)Bon

eno

dules:form

edby

cells

from

allsegments

(2)Cho

ndrogenicpo

tential:occurred

incells

from

allsegments

WJ-MSC

sareagood

cell

source

forautologous/

allogeneicstem

cellsource.

4.Batsalietal.

2017

[83]

Invitro

(i)Bon

emarrow

mesenchym

alstem

cell(BM-M

SCs)

(ii)Wharton

’sjelly

Differentiationmedium:

Osteogenicmedium:

(1)High-glucoseDMEM

(2)10%

serum

(3)10

−7M

dexamethasone

(i)Explant

metho

d(ii)Partof

UC:

maternal,middle,

andfetal

segm

ents

(1)Osteogenicdifferentiation(A

lizarin

Red

andvonKossa

staining):WJ-

MSC

sshow

edsimilarstaining

potentialasBM-M

SC(2)Real-time-

(RT-)PCR:

(i)Osteocyte-related

gene

expression

:higherexpression

ofRun

t-related

transcriptionfactor

2(RUNX2),distal-lessho

meoboxprotein5(D

LX5),

The

osteogenic

differentiationpo

tentialo

fWJ-MSC

sisregulatedby

WISP1andsFRP4,

respectively.


Table1:Con

tinu

ed.

No.

Autho

rand

year

Typeof

stud

ySubject/sample

Indu

ctionfactor

WJ-MSC

isolation

metho

dResults

Con

clusion

mesenchym

alstem

cells

(WJ-MSC

s)(4)25

μg/mLL-ascorbicacid

(5)3mM

NaH

2PO4

osteocalcin(O

CN),andalkalin

eph

osph

atase(A

LP)by

BM-M

SCs

(3)Differentialexpression

ofWNTligands;sFR

P4(secretedfrizzled-related

protein4)

andWISP1(W

NT1-indu

ciblesignallin

gpathway

protein1)

weresignificantlyredu

cedin

WJ-MSC

s(4)WISP1im

plicated

inosteogenicdifferentiation;

RUNX2,ALP

,and

OSC

weresignificantlyup

regulated

5.Zajdeletal.

2017

[84]

Invitro

(i)Adipo

setissue

(AT-

MSC

s)(ii)WJ-MSC

s

Osteogenicmedium

(Lon

za):

(1)Dexam

ethasone

(2)Ascorbicacid

(3)β-G

lyceroph

osph

ate

(i)Com

mercialAT-

MSC

sandUC-

MSC

s(ii)Partof

UC:n

otmention

ed

(1)Calcium

depo

sition

byAlizarin

Red

staining

(i)Calcium

depo

sition

was

greaterin

AT-M

SCswhencomparedto

WJ-

MSC

s(2)ALP

activity

(i)ALP

activity

was

higher

inAT-M

SCswhencomparedto

WJ-MSC

s(3)Osteoprotogerin

(OPG)secretion

(i)Bothosteo-indu

cedcelltypesshow

edhigh

OPGsecretionwhen

comparedto

control

(4)Osteocalcin

(OC)secretion

(i)OCsecretionwas

higher

inWJ-MSC

swhencomparedto

AT-M

SCs

WJ-MSC

shave

theability

todifferentiateinto

the

osteogeniclin

eage.

6.Mechiche

Alamietal.

2017

[88]

Invitro

(i)WJ-MSC

sCalcium

phosph

ate(CaP)substratebu

ild-

up(w

itho

utosteogenicindu

ction

(i)Com

mercialUC-

MSC

s(ii)Partof

UC:n

otmention

ed

(1)Geneexpression

analysis

(i)Day

7:Run

t-relatedtranscriptionfactor

2(RUNX2)

andsecreted

phosph

oprotein

1(SPP-1)wereup

regulated

(ii)Day

14:collagentype

1Alpha

1(COL1

A1)

andALP

wereup

regulated

(iii)

Day

21:bon

egamma-carboxyglutamicacid-con

tainingprotein(BGLP

)andALP

wereup

regulated

(2)Nod

ulecharacterization

(i)Hem

atoxylin-eosin-saff

ron(H

ES)

staining

revealed

continuo

uslayersof

cells

atthesurfaceof

theno

dulewithrand

omlydistribu

tedcells

embedd

edwithinfibrou

stissues

(ii)Masson’strichrom

estaining

show

edthepresence

ofgreen-stained

fibrou

stissue

compo

sedof

newlyform

edcollagen

Excellent

osteogenic

potentialofsprayed

CaP

and

WJ-MSC

sin

bone

tissue

engineering

7.Szepesietal.

2016

[61]

Invitro

(i)WJ-MSC

s(ii)AT-M

SCs

(iii)

Periodo

ntal

ligam

entMSC

s(PDL-MSC

s)


(i)Stem

Pro

OsteogenesisDifferentiation

kit

(ii)Stem

Pro

Cho

ndrogenesis

Differentiationkit

(iii)

Stem

Pro

Adipo

genesisDifferentiation

kit

(iv)

End

othelialC

ellG

rowth

Medium

(i)Enzym

atic

digestionUC-

MSC

s(ii)Partof

UC:n

otmention

ed

(1)Osteogenicdifferentiation:

AT-M

SCsandPDL-MSC

sshow

edgreater

calcium

depo

sition

(2)Osteogenicdifferentiationassessed

viaRT-PCR:

(i)Run

t-relatedtranscriptionfactor

2(RUNX2):allcelltypesshow

edhigh

expression

afterindu

ction

(ii)Alkalineph

osph

atase(A

LP):AT-M

SCsandPDL-MSC

sshow

edincreasedexpression

,but

ALP

was

significantlylower

inWJ-MSC

s(iii)

Calcium

depo

sition

:AT-M

SCsandPDL-MSC

sshow

edgreatercalcium

depo

sition

ascomparedto

WJ-MSC

s(3)There

was

asignificant

correlationbetweenCD90

expression

and

thelevelsof

calcium

depo

sition

indifferentMSC

isolates

WJ-MSC

shave

osteogenic

potentialand

aregood

cell

sourcesforbone

regeneration

.

8.Lim

etal.

2016

[79]

Invitro

Hum

anWJ-MSC

s(i)Fetalsegment

(ii)Maternalsegment

(iii)

Middlesegm

ent

(i)Osteogenicmedium

(1)Alpha-M

EM

(2)Dexam

ethasone

(3)Ascorbicacid

(4)β-G

lycerolp

hosphate

(ii)Adipo

genicmedium:

(1)DMEM/F12

(2)3-Isobutyl-3-m

ethylxanthine

(3)Dexam

ethasone

(4)Insulin

(iii)

Cho

ndrogenicmedium

(1)Alpha-M

EM

(i)Enzym

atic

digestion

(ii)Maternal,middle,

andfetal

segm

ents

Bon

eno

dules:form

edby

cells

from

allsegments

WJ-MSC

sareagood

cell

source

forbone

regeneration

.


Table1:Con

tinu

ed.

No.

Autho

rand

year

Typeof

stud

ySubject/sample

Indu

ctionfactor

WJ-MSC

isolation

metho

dResults

Con

clusion

(2)Transform

inggrow

thfactor

3(TGF-β3)

9.Kargozar

etal.2018

[80]

Invitro

and

invivo

(i)BM-M

SCs

(ii)AT-M

SCs

(iii)

UC-M

SCs

(i)Nanocom

posite

scaffolds

(3D

bioactive

glass/gelatinscaffolds

(BaG

/Gel)

consisting

ofSiO2-P2O

5-CaO

(64%

SiO2,5%

P2O

5,and31%

CaO

).

(i)Enzym

atic

digestion

(ii)Partof

UC:n

otmention

ed

Invitrostud

y(1)Cellviability:

(i)Scaffoldhadno

significant

inhibitory

effecton

MSC

proliferation

over

time

(ii)MSC

proliferation

gradually

increasedwithincubation

time

BM-M

SCs,grow

non

BaG

/Gelnano

compo

site

scaffolds,are

possible

sourcesforbone

regeneration

.

Invivo

stud

y(1)Histologicalo

bservation

s:(i)H&Estaining:allMSC

-seededscaffolds

successfullygeneratednew

bone

anddemon

stratedan

ongoinghealingprocessat

4and12

weeks

aftertransplantation.

The

UC-M

SC-seededscaffoldshow

edsignificantlyincreasedneovascularization

comparedto

theothers

(ii)IH

Cstaining:increased

expression

ofOCN

andALP

intheBM-M

SC-

seeded

scaffold.

Vascularendo

thelialgrowth

factor

(VEGF)

expressed

inallthe

grou

psof

treatedcell/scaffold.

Increasedneovascularization

withtheUC-M

SC-seededscaffold

(iii)

Histomorph

ometry:B

M-M

SC-seededscaffolds

show

edmorebone

regeneration

at4and12

weeks

10.

Tod

eschi

etal.2015

[81]

Invivo

(i)UC-M

SCs

(ii)BM-M

SCs

(i)Ceram

icscaffolds

(Skelite;

4×4×4mm

cubesof

33%

hydroxyapatiteand67%

silicon

-stabilizedtricalcium

phosph

ate,Si-

TCP)

(ii)Platelet-rich

plasma(PRP)

(iii)

Con

dition

edmedium

(CM)

(i)Explant

metho

d(ii)Partof

UC:n

otmention

ed

(1)Histologicalassessm

entrevealed

theform

ationan

immaturebone-like

structures

andcompactfibrou

stissue

inUC-M

SC-seededconstructs

(2)Polarized

light

exam

inationrevealed

lessorganizedcollagenfibersin

the

UC-M

SC-seededscaffolds.T

heim

maturebone-likematrixin

UC-M

SC-

seeded

scaffolds

was

mostly

filledwithalooseconn

ective

tissue

(3)Histologicalevaluationin

anorthotop

icmou

semod

elshow

edthatno

neof

thebone

defectshadcompletelyclosed.H

owever,goldMTCstaining

indicatedthepresence

ofredbloodcells

inbloodvessel-likestructures

which

issignificant

intheUC-M

SC-transplantedgrou

p(4)Osteocyteswereclearlydetectablein

theBMMSC

-seededscaffolds

(5)How

ever,hum

anALU

sequ

enceswereno

tdetectedin

osteocytes

within

thenewlyform

edbone

intheUC-M

SCim

plantsandno

nseeded

implants

UC-M

SCsprom

otebone

regeneration

.

11.

Karadas

etal.2014

[74]

Invitro

(i)WJ-MSC

s(ii)BM-M

SCs

(iii)

Menstrualblood

mesenchym

alstem

cells

(MBMSC

s)

(i)Collagenscaffolds

within

situ

calcium

phosph

ate(CaP)

(ii)Differentiationmedium:

(1)High-glucoseDMEM

(2)10

nMdexamethasone

(3)50

μg/mLascorbicacid

(4)10

mM

β-glyceroph

osph

ate

(5)10%

FBS

(6)100un

its/mLpenicillin

(7)100μg/mLstreptom

ycin

(i)Explant

metho

d(ii)Partof

UC:n

otmention

ed

(1)Cellp

roliferationassays:

(i)WJ-MSC

son

tissue

cultu

repo

lystyrene(TCPS)

andcollagenwitho

utCaP

treatm

entincreasedproliferation

(2)Cellattachm

ent(fluo

rescence

staining):

(i)Goodattachmentof

both

celltypesto

thescaffold

(ii)Con

focalm

icrographs

show

edthatthecells

wereableto

penetrateinto

thepo

res


(i)ALP

assay:

(a)WJ-MSC

sshow

edbetter

differentiationon

untreatedandCaP-

containing

foam

sthan

ingrow

thmedium

(b)ALP

levelswerehigherin

WJ-MSC

sgrow

non

CaP-freefoam

sthan

onTCPS

(c)ALP

activity

was

significantlyhigher

incells

grow

non

collagenwith

CaP

crystalsform

edin

situ

forboth

WJ-MSC

sandMBMSC

s(days14

and21)

Collagenfoam

withtheuse

ofCaP

crystalsform

edin

situ

enhances

theosteogenic

indu

ctionof

WJ-MSC

s.

vonKossa

staining:

(1)WJ-MSC

shadhigher

ALP

activity

anddenser

mineraldepo

sition

comparedto

MBMSC

s


Table1:Con

tinu

ed.

No.

Autho

rand

year

Typeof

stud

ySubject/sample

Indu

ctionfactor

WJ-MSC

isolation

metho

dResults

Con

clusion

12.

Ram

esh

etal.2014

[93]

Invitro

(i)WJ-MSC

s

(i)Hydrogelalginatemicrospheres

(ii)Osteogenicdifferentiationmedia:

(1)Basalmedium

(2)10

mM

β-glyceroph

osph

ate

(3)1mM

dexamethasone

(4)5mg/mLascorbicacid

(i)Explant

metho

d(ii)Partof

UC:n

otmention

ed

(1)Characterizationof

osteod

ifferentiated

WJ-MSC

svia:

(i)Bradfordassay:calcium

depo

sition

increasedin

2%alginate

(ii)Alizarin

Red

staining:excellent

matrixmineralizationin

WJ-MSC

sim

mobilizedon

2%alginate

(iii)

Immun

ocytochemicalanalysis(osteocalcin):significant

expression

ofosteocalcinin

WJ-MSC

aggregates

in1.5%

and2%

alginateat

day21

(2)Genotypicanalysisof

encapsulated

WJ-MSC

sshow

edthat

OCN

andRun

x2wereup

regulatedin

1.5%

and2%

alginate

WJ-MSC

sencapsulated

inhydrogelalginate

microsphereshave

osteogenicpo

tentialfor

stem

cell-basedtissue

engineering.

13.

Babaetal.

2012

[16]

Invivo

&in

vitro

Hum

anum

bilicalcord

mesenchym

alstem

cells

(hUC-M

SCs)


(i)NH

OsteoDiff

Medium

(ii)NH

Adipo

Diff

Medium

(iii)

rhBMP2

(iv)

Scaffold

(i)Enzym

atic

digestion

(ii)Partof

UC:n

otmention

ed


strong

calcium

depo

sition

Adipo

genicdifferentiation:

lipid

drop

letprod

uction

(4)H&Estaining:p

ositiveforbone

tissue

prod

ucingosteocalcin(O

CN)

(5)RT-PCR:h

ighexpression

ofRUNX2,ALP

,and

OCN

ascomparedto

undifferentiated

cells

UC-M

SCssupp

lemented

withgrow

thfactorsand

serum

have

osteogenic

differentiationpo

tentialfor

bone

regeneration

.

14.

Penolazzi

etal.2012

[89]

Invitro

(i)WJ-MSC

s(i)Porcine

urinarybladdermatrix(pUBM)

(i)Enzym

atic

digestion

(ii)Partof

UC:n

otmention

ed

(1)Proliferationassays

show

ed(i)pU

BM

didno

thave

aneffecton

cells

insuspension

cond

itions

but

affectedcells

cultu

redin

adherent

cond

itions

(ii)Viablecells

wereho

mogenou

slydistribu

tedover

theentire

scaffold

(2)TUNELassays

show

ed(i)Noapop

tosisin

hWJ-MSC

scultu

redin

agarose-coated

wellswith

increasing

amou

ntsof

pUBM

(ii)The

scaffoldup

regulatedcyclin

D1

(iii)

Matrixmetanop

roteinase(M

MPI3)w

aslowerbu

tthisdidno

taffect

β-catenin

(3)Morph

ologicalcharacterization

:(i)Scanning

electron

microscop

e(SEM)analysisshow

ed(a)There

was

asignificant

interactionbetweenthecells

andthe

biom

aterial

(b)WJ-MSC

scompletelywereenvelopedin

pUBM

particlesto

form

smooth

spheroids,displaying

anECM

networkcovering

thesurface

(c)The

cells

andthebiom

aterialformed

adensestructure

(ii)X-ray

energy-dispersivespectroscopy

(EDX)show

edthatspheroids

inosteogenicmedium

containedhigh

amou

ntsof

calcium

and

phosph

orou

s(highdegree

ofmineralization)

(iii)

Transmission

electron

microscop

y(TEM)show

edthepresence

offocalcon

tactsbetweencells

andthepU

BM

scaffold

(4)RT-PCR:

(i)RUNX2expression

was

notaffectedby

pUBM,but

increasedup

onosteoind

uction

(ii)WJ-MSC

sseeded

onpU

BM

wereableto

prod

uceCol

IAIandOPN

after21

days

inosteogenicmedium

(iii)

WJ-MSC

sshow

edincreasedALP

activity

andability

todepo

sit

mineralized

matrix

(iv)

OPN

expression

was

higher

incells

grow

non

pUBM

scaffolds

inosteogenicmedium

than

inosteogenicmedium

alon

e

The

combination

ofWJ-

MSC

sandpU

BM

show

sthe

prom

iseof

scaffolds

forbone

regeneration

.

15.

Wangetal.

2011

[94]

Invitro

(i)UC-M

SCs

(i)Poly-L-lacticacid

(PLL

A)scaffold

(ii)Osteogenicindu

ctionmedium:

(1)Lo

w-glucose

DMEM

(2)10%

FBS

(3)1%

penicillin/streptom

ycin

(4)100nM

dexamethasone,

(5)10

mM

sodium

β-

glycerop

hosphate,

(i)Enzym

atic

digestion

(ii)Partof

UC:n

otmention

ed

(1)Biochem

icalassays

wereperformed

toassess

(i)DNAcontent:osteogenicpartsof

theC-cell-Ocompo

siteshadhigher

DNAcontentsthan

theO-O

compo

sitesandtheosteogenicpartsof

theC-O

compo

sites

(ii)Glycosaminoglycan(G

AG)content:allosteogenicgrou

pshadsimilar

GAGcontents

(iii)

Hydroxyproline(H

YP)content:osteogenicgrou

pshadasignificantly

higher

HYPcontent

WJ-MSC

sareasuitablecell

source

forasand

wich

approach

strategy

inosteocho

ndraltissue

engineering.


Table1:Con

tinu

ed.

No.

Autho

rand

year

Typeof

stud

ySubject/sample

Indu

ctionfactor

WJ-MSC

isolation

metho

dResults

Con

clusion

(6)50

μg/mLascorbicacid

2-ph

osph

ate(A

A2P

)(7)10

nM1α

,25-dihydroxyvitam

inD3

(i)Cho

ndrogenicindu

ction

medium:

(1)High-glucoseDMEM

(DMEM-

HG)

(2)1%

nonessentialam

inoacids

(NEAA)

(3)1x

sodium

pyruvate

(4)1x

insulin

-transferrin-selenium

prem

ix(ITS)

(5)50

μg/mL(A

A2P

)(6)40

μg/mLL-proline

(7)100nM

dexamethasone

(8)10

ng/m

LTGF-β1

(iv)

Calcium

content:osteogenicgrou

psshow

edsignificantlyincreased

calcium

levelsover

time

(2)Histologicalanalyses:po

sitive

Alizarin

Red

staining

inthe

osteogenicgrou

p(3)RT-PCR:

(i)Collagentype

II(ColII):no

texpressedin

theosteogenicgrou

p(ii)Collagentype

I(ColI):u

pregulated

inallgroup

s(iii)

RUNX2:increasedin

theosteogenicgrou

p(iv)

Aggrecan:

increasedsignificantlyin

chon

drogenicgrou

p

16.

Schn

eider

etal.2010

[85]

Invitro

Hum

anmesenchym

alstem

cells

(hMSC

):(i)UC-M

SCs

(ii)BM-M

SCs

(i)Scaffold:

3Dcollagengel

(ii)Osteogenicindu

ctionmedium:

(1)Lo

w-glucose

DMEM

(2)10%

FCS

(3)100nM

dexamethasone

(4)10

mM

sodium

β-

glycerop

hosphate

(5)0.05

mM/L-ascorbicacid

2-ph

osph

ate

(iii)

Adipo

genicindu

ctionmedium:

(1)DMEM

high

glucose

(2)1μM

dexamethasone

(3)0.2mM

indo

methacin

(4)0.01

mg/mLinsulin

(5)0.5mM

3-isobutyl-1-

methylxanthine

(6)10%

FCS

(i)Enzym

atic

digestion

(ii)Partof

UC:n

otmention

ed

(1)Scaffold:

(i)3D

collagengelu

nderwentprogressivecontraction

(ii)After

osteogenicdifferentiation,

collagengelswerestronger

and

harder

withBM-M

SCsandUC-M

SCs

(2)Characterizationof

BM-M

SCsandUC-M

SCsby

(i)Osteogenicdifferentiation:

UC-M

SCsshow

edincreasedextracellular

matrix(ECM)depo

sition

byAlizarin

Red

(AR)staining

(ii)Adipo


UC-M

SCsshow

edalim

ited

numberof

smalllipid

vacuoles

stainedby

OilRed

O(O

RO)

(iii)

Immun

ofluo

rescence:positiveexpression

ofcollagenIV

andlaminin

inUC-M

SCsafter21

days

ofosteogenicdifferentiationon

collagen

gels

(iv)

Immun

ohistochem

istryanalyses:p

ositiveexpression

ofosteop

ontin

(OPN)andbispho

spho

nate

[2-(2-pyridinyl)ethylid

ene-BP](PEBP)

inthecollagengel

(3)TEM

analysis:

(i)Osteogenicdifferentiation:

contractionof

thecollageno

usmatrixon

the

UC-M

SC-seededcollagensurface

(ii)Adipo


UC-M

SC-derived

lipid

vacuoles

weresm

all

andstainedwithtoluidineblue

(4)RT-PCR:U

C-M

SCsexpressedcollagenI,collagenIII,collagenIV

,andlaminin

(5)Cellm

igration

:migrating

UC-M

SCsappeared

asspindle-shaped

cells

withelon

gatedcytoplasmicprocesses

UC-M

SCshave

asignificant

therapeuticim

pactin

bone

tissue

engineeringin

the

future.

17.

Hsieh

etal.

2010

[14]

(i)In

vitro

(i)WJ-MSC

s(ii)BM-M

SCs

Osteogenicdifferentiationmedium:

(1)DMEM

(2)10%

FBS

(3)0.1mM

dexamethasone

(4)10

mM

β-glyceroph

osph

ate

(5)50

mM

ascorbicacid

(i)Enzym

atic

digestion

(ii)Partof

UC:n

otmention

ed

(1)Array

data

show

edthat

both

BM-M

SCsandWJ-MSC

sexpressed

multilin

eage

differentiationprop

erties.

(2)Real-time-PCR:

BM-M

SCs>WJ-MSC

sin

term

sof

(i)Adipo

cyticmarkerexpression

:lipoprotein

lipase(LPL),leptin,

peroxisomeproliferator-activatedreceptor

gamma(PPARγ),and

fattyacid-binding

protein4(FABP4)

(ii)Lipidaccumulation

(3)Osteogenicdifferentiationpo

tential

(i)BM-M

SCs>WJ-MSC

swhencultu

redin

definedMesenCult

(ii)Expressed

higher

levelsof

ALP

,SPP-1,and

RUNX2

WJ-MSC

sarecapableof

differentiatinginto

the

osteogeniclin

eage,but

BM-

MSC

saresuperior.


Table1:Con

tinu

ed.

No.

Autho

rand

year

Typeof

stud

ySubject/sample

Indu

ctionfactor

WJ-MSC

isolation

metho

dResults

Con

clusion

(iii)

BM-M

SCs>WJ-MSC

swhencultu

redin

definedcultured

in10%

FBS:

(a)Expressed

higher

levelsof

ALP

,osteopo

ntin,and

RUNX2

(4)ALP

staining

(betterosteogenicability)

18.

Hou

etal.

2009

[15]

(i)In

vitro

(i)hU

C-M

SCs

(ii)BM-M

SCs

(i)BMP-2

treatm

ent:

(a)Bon

emorph

ogeneticprotein2-

blocking

antibodies

(BMP2Ab)

(b)Recom

binant

human

BMP2

(rhB

MP2)

(c)Noggin

(ii)Osteogenicdifferentiationmedium:

(1)DMEM/F12

(2)10%

FBS

(3)Dexam

ethasone

(4)Ascorbicacid

(AsA

)2-

phosph

ate

(5)β-G

lyceroph

osph

ate

(iii)

Adipo

genicdifferentiation

medium:

(1)DMEM/F12

(2)1%

FBS

(3)100nM

dexamethasone

(4)1nM

insulin

(iv)

Cho

ndrogenicdifferentiation

medium:

(1)DMEM/F12

(2)Dexam

ethasone

(3)AsA

(4)TGF-β1

(5)ITS+

Premix

(i)Enzym

atic

digestion

(ii)Partof

UC:n

otmention

ed

(1)Trilin

eage

differentiation

(i)Osteogenesis:osteon

ectin,

ALP

,and

RUNX2wereexpressed

(ii)Cho

ndrogenesis:COLII,collagentype

X(COLX),andaggrecan

were

expressed

(iii)

Adipo

genesis:adipsin,

PPARγ,andlip

oprotein

lipase

(2)ALP

activity

was

significantlyincreasedin

BMP2-indu

cedUC-

MSC

s(i)Western

blot

revealed

activation

oftheBMP2signalingpathway

inboth

celltypesviaSM

ADs,p38,andextracellularregulatedkinase

activation

BMP2-indu

cedUC-M

SCs

have

good

osteogenic

differentiation(ind

icated

bytheactivation

ofBMP2

signaling)

andmay

beused

intissue-engineeredbone.


MSCs were found to be superior to WJ-MSCs in terms ofdifferentiating into the osteogenic lineage after 21 dayscompared. In a different study by Lim and colleagues,MSCs derived from different parts of the umbilical cord,i.e., the fetal, middle, and maternal segments, have theability to differentiate into osteogenic lineage cells usingthe osteogenic medium consisting of dexamethasone,ascorbic acid, and β-glycerophosphate. The fetal part wasshown to have the best differentiation potential [79]. Astudy by Hsieh et al. [14] showed that BM-MSCs expressmore osteogenic genes compared to WJ-MSCs; conversely,WJ-MSCs are more responsible for angiogenesis [66].Bone morphogenetic protein 2 (BMP-2) was used in astudy by Hou et al. to promote osteogenic differentiationin WJ-MSCs [15].

4.2.2. Physical Induction. It is noteworthy that the microenvi-ronment influences cell behaviour and leads to the pro-duction of a specific chemical composition that buildsthe ECM. Therefore, fabricated scaffolds have been activelyinvestigated to find better materials and to produce thebest structure of ECM-like components. From the databasesearch, eight out of 18 articles investigated the fabricationof various scaffolds to test the potential of WJ-MSCs topromote complex bone regeneration. Various biomaterialswere used to construct these scaffolds, ranging from colla-gen hydrogels [85] to bioactive glass [80]. 3D scaffoldshave been documented as one of the best carriers for celldelivery in bone regeneration. The ideal scaffold shouldbe osteoconductive, biocompatible, and bioresorbable; pos-sess interconnected porosity; and promote cell binding/attachment [82, 86].

The first phase in scaffold development used collagen asthe main organic component in bone tissue for bone grafting[85]. Collagen type I can be isolated from Sprague-Dawley rattails after processing and pelleting. Genipin has been selec-tively used for crosslinking collagen scaffolds to improvethe stability and mechanical strength of the scaffolds in theculture medium [74]. Other crosslinkers have also been used,such as glutaraldehyde and formaldehyde. However, thosehave been reported to have some cytotoxic effects [87]. Cal-cium phosphate is another major constituent in bone thathas been widely studied as a scaffold material for bone tissueengineering [88]. A study by Karadas et al. [74] produced anin situ mineralized collagen scaffold whereby, after crosslink-ing with genipin, the scaffold was immersed in a calcium andphosphate solution. As a result, highly integrated calciumphosphate minerals were successfully formed [74]. The com-bination of collagen I and III has also been reported to resem-ble the native ECM, where umbilical cord MSCs (UC-MSCs)were found to have better osteogenic potential compared toBM-MSCs [85]. In addition, porcine ECM, derived fromthe urinary bladder, has also been used as a biomaterial forscaffold preparation as it contains collagen, glycoproteins,glycosaminoglycans, and GFs [89].

Scaffold design then moved to the second phase, inwhich bioactive glass has been used as a scaffold in bonetissue engineering [80]. Kargozar and colleagues used acombination of bioactive glass/gelatin (BaG/Gel) scaffolds,

aiming for a highly porous structure, which is consideredideal for bone substitution. A comprehensive physiochem-ical analysis showed that the structure had an intact, 3Dporous microstructure with interconnected pores. It wasalso shown that the properties were very close to thoseof natural spongy bone [80]. They demonstrated that neo-vascularization was significantly better in the UC-MSC-seeded scaffold when compared to the BM-MSC-seededscaffold, indicating that the BaG/Gel scaffold is MSCtype-dependent. A study by Todeschi and colleagues usedhydroxyapatite (HA), beta-tricalcium phosphate (β-TCP),or a mixture of the two [81] as the scaffold. They showedthat a significantly higher number of blood vessels werepresent in the UC-MSC-seeded implants [81].

5. Conclusion

WJ-MSCs were first isolated by Mitchell et al. in 2003.During embryogenesis, totipotent cells such as primordialgerm cells and hematopoietic stem cells migrate from theyolk sac through this region to populate target tissues inthe embryo and fetus [90]. Characterization indicated thatthese cells are stem cells, as they express c-kit and can dif-ferentiate into neural cells. WJ-MSCs have similar prolifer-ation and differentiation capacity and have multilineagedifferentiation potential [77], including osteogenesis. Thisreview demonstrates that WJ-MSCs are capable of differ-entiation into osteoblasts, which may be useful for moreeffective bone fracture healing as these cells have beenshown to migrate into and colonize a collagenous matrix.With the aid of 3D scaffolds, cell proliferation and survivalare improved as these scaffolds provide structural stabilitysimilar to that of bone. However, MSCs have to be com-patible with the scaffold prior to integration and incorpo-ration into engineered bone.

Conflicts of Interest

The authors declare that they have no conflicts of interest.

Acknowledgments

The research was carried out with the financial support ofUniversiti Kebangsaan Malaysia and AMRUS Medik Sdn.Bhd. through research grant numbers FF-2017-482 andFF-2017-020, respectively.

References

[1] M. L. Weiss and D. L. Troyer, “Stem cells in the umbilicalcord,” Stem Cell Reviews, vol. 2, no. 2, pp. 155–162, 2006.

[2] L. Raio, F. Ghezzi, E. di Naro et al., “Prenatal diagnosis of alean umbilical cord: a simple marker for the fetus at risk ofbeing small for gestational age at birth,” Ultrasound in Obstet-rics & Gynecology, vol. 13, no. 3, pp. 176–180, 1999.

[3] E. Di Naro, F. Ghezzi, L. Raio, M. Franchi, and V. D’Addario,“Umbilical cord morphology and pregnancy outcome,” Euro-pean Journal of Obstetrics & Gynecology and ReproductiveBiology, vol. 96, no. 2, pp. 150–157, 2001.


[4] F. A. Meyer, Z. Laver-Rudich, and R. Tanenbaum, “Evidencefor a mechanical coupling of glycoprotein microfibrils withcollagen fibrils in Wharton’s jelly,” Biochimica et BiophysicaActa (BBA) - General Subjects, vol. 755, no. 3, pp. 376–387,1983.

[5] I. B. Copland, S. L. Adamson, M. Post, S. J. Lye, andI. Caniggia, “TGF-β3 expression during umbilical cord devel-opment and its alteration in pre-eclampsia,” Placenta,vol. 23, no. 4, pp. 311–321, 2002.

[6] M. Mizoguchi, Y. Suga, B. Sanmano, S. Ikeda, and H. Ogawa,“Organotypic culture and surface plantation using umbilicalcord epithelial cells: morphogenesis and expression of differ-entiation markers mimicking cutaneous epidermis,” Journalof Dermatological Science, vol. 35, no. 3, pp. 199–206, 2004.

[7] J. E. Davies, J. T. Walker, and A. Keating, “Concise review:Wharton’s jelly: the rich, but enigmatic, source of mesenchy-mal stromal cells,” Stem Cells Translational Medicine, vol. 6,no. 7, pp. 1620–1630, 2017.

[8] K. Sobolewski, E. Bańkowski, L. Chyczewski, and S. Jaworski,“Collagen and glycosaminoglycans ofWharton’s jelly,”Neona-tology, vol. 71, no. 1, pp. 11–21, 1997.

[9] H.-S. Wang, et al.S.-C. Hung, S.-T. Peng et al., “Mesenchymalstem cells in the Wharton’s jelly of the human umbilical cord,”Stem Cells, vol. 22, no. 7, pp. 1330–1337, 2004.

[10] I. Kalaszczynska and K. Ferdyn, “Wharton’s jelly derived mes-enchymal stem cells: future of regenerative medicine? Recentfindings and clinical significance,” BioMed Research Interna-tional, vol. 2015, Article ID 430847, 11 pages, 2015.

[11] M. Dominici, K. le Blanc, I. Mueller et al., “Minimal criteria fordefining multipotent mesenchymal stromal cells. The Interna-tional Society for Cellular Therapy position statement,”Cytotherapy, vol. 8, no. 4, pp. 315–317, 2006.

[12] S. Tipnis, C. Viswanathan, and A. S. Majumdar, “Immunosup-pressive properties of human umbilical cord-derived mesen-chymal stem cells: role of B7-H1 and IDO,” Immunology &Cell Biology, vol. 88, no. 8, pp. 795–806, 2010.

[13] X. Y. Wang, Y. Lan, W. Y. He et al., “Identification of mesen-chymal stem cells in aorta-gonad-mesonephros and yolk sac ofhuman embryos,” Blood, vol. 111, no. 4, pp. 2436–2443, 2008.

[14] J.-Y. Hsieh, Y.-S. Fu, S.-J. Chang, Y.-H. Tsuang, and H.-W. Wang, “Functional module analysis reveals differentialosteogenic and stemness potentials in human mesenchymalstem cells from bone marrow and Wharton’s jelly of umbilicalcord,” Stem Cells and Development, vol. 19, no. 12, pp. 1895–1910, 2010.

[15] T. Hou, J. Xu, X. Wu et al., “Umbilical cord Wharton’s jelly: anew potential cell source of mesenchymal stromal cells forbone tissue engineering,” Tissue Engineering Part A, vol. 15,no. 9, pp. 2325–2334, 2009.

[16] K. Baba, Y. Yamazaki, S. Ikemoto, K. Aoyagi, A. Takeda, andE. Uchinuma, “Osteogenic potential of human umbilicalcord-derived mesenchymal stromal cells cultured with umbil-ical cord blood-derived autoserum,” Journal of Cranio-Maxillofacial Surgery, vol. 40, no. 8, pp. 768–772, 2012.

[17] G. la Rocca, M. Lo Iacono, T. Corsello, S. Corrao, F. Farina,and R. Anzalone, “Human Wharton’s jelly mesenchymal stemcells maintain the expression of key immunomodulatory mol-ecules when subjected to osteogenic, adipogenic and chondro-genic differentiation in vitro: new perspectives for cellulartherapy,” Current Stem Cell Research & Therapy, vol. 8,no. 1, pp. 100–113, 2013.

[18] M. Esposito, A. Lucariello, C. Costanzo et al., “Differentiation ofhuman umbilical cord-derived mesenchymal stem cells, WJ-MSCs, into chondrogenic cells in the presence of pulsed elec-tromagnetic fields,” In Vivo, vol. 27, no. 4, pp. 495–500, 2013.

[19] M.Messerli, A.Wagner, R. Sager et al., “Stem cells from umbil-ical cord Wharton’s jelly from preterm birth have neuroglialdifferentiation potential,” Reproductive Sciences, vol. 20,no. 12, pp. 1455–1464, 2013.

[20] Y. B. Cui, S. S. Ma, C. Y. Zhang et al., “Human umbilical cordmesenchymal stem cells transplantation improves cognitivefunction in Alzheimer’s disease mice by decreasing oxidativestress and promoting hippocampal neurogenesis,” BehaviouralBrain Research, vol. 320, pp. 291–301, 2017.

[21] I. Garzón, M. A. Martín-Piedra, C. Alfonso-Rodríguez et al.,“Generation of a biomimetic human artificial cornea modelusing wharton’s jelly mesenchymal stem cells,” InvestigativeOpthalmology & Visual Science, vol. 55, no. 7, pp. 4073–4083, 2014.

[22] J. Hu, Y. Wang, H. Gong et al., “Long term effect and safety ofWharton’s jelly-derived mesenchymal stem cells on type 2 dia-betes,” Experimental and Therapeutic Medicine, vol. 12, no. 3,pp. 1857–1866, 2016.

[23] M. L. Weiss, et al.C. Anderson, S. Medicetty et al., “Immuneproperties of human umbilical cord Wharton’s jelly-derivedcells,” Stem Cells, vol. 26, no. 11, pp. 2865–2874, 2008.

[24] F. Granero-Moltó, J. A. Weis, M. I. Miga et al., “Regenerativeeffects of transplanted mesenchymal stem cells in fracturehealing,” Stem Cells, vol. 27, no. 8, pp. 1887–1898, 2009.

[25] H. Kaneki, R. Guo, D. Chen et al., “Tumor necrosis factor pro-motes Runx2 degradation through up-regulation of Smurf1and Smurf2 in osteoblasts,” Journal of Biological Chemistry,vol. 281, no. 7, pp. 4326–4333, 2006.

[26] X. Zhou, et al.Z. Zhang, J. Q. Feng et al., “Multiple functions ofOsterix are required for bone growth and homeostasis in post-natal mice,” Proceedings of the National Academy of Sciences ofthe United States of America, vol. 107, no. 29, pp. 12919–12924, 2010.

[27] Y.-i. Kawabe, Y. X. Wang, I. W. McKinnell, M. T. Bedford, andM. A. Rudnicki, “Carm1 regulates Pax7 transcriptional activitythrough MLL1/2 recruitment during asymmetric satellite stemcell divisions,” Cell Stem Cell, vol. 11, no. 3, pp. 333–345, 2012.

[28] S. J. Prasanna, D. Gopalakrishnan, S. R. Shankar, and A. B.Vasandan, “Pro-inflammatory cytokines, IFNγ and TNFα,influence immune properties of human bone marrow andWharton jelly mesenchymal stem cells differentially,” PLoSOne, vol. 5, no. 2, article e9016, 2010.

[29] T. Deuse, M. Stubbendorff, K. Tang-Quan et al., “Immunoge-nicity and immunomodulatory properties of umbilical cordlining mesenchymal stem cells,” Cell Transplantation, vol. 20,no. 5, pp. 655–667, 2011.

[30] R. Sarugaser, D. Lickorish, D. Baksh, M. M. Hosseini, and J. E.Davies, “Human umbilical cord perivascular (HUCPV) cells: asource of mesenchymal progenitors,” Stem Cells, vol. 23, no. 2,pp. 220–229, 2005.

[31] S. B. Goodman, P. Huie, Y. Song et al., “Cellular profile andcytokine production at prosthetic interfaces: study of tissuesretrieved from revised hip and knee replacements,” The Jour-nal of Bone and Joint Surgery, vol. 80, no. 3, pp. 531–539, 1998.

[32] N. M. Moll and R. M. Ransohoff, “CXCL12 and CXCR4 inbone marrow physiology,” Expert Review of Hematology,vol. 3, no. 3, pp. 315–322, 2010.


[33] E. C. Butcher and L. J. Picker, “Lymphocyte homing andhomeostasis,” Science, vol. 272, no. 5258, pp. 60–67, 1996.

[34] L. da Silva Meirelles, A. M. Fontes, D. T. Covas, and A. I.Caplan, “Mechanisms involved in the therapeutic propertiesof mesenchymal stem cells,” Cytokine & Growth FactorReviews, vol. 20, no. 5-6, pp. 419–427, 2009.

[35] S. Zwingenberger, Z. Yao, A. Jacobi et al., “Enhancement ofBMP-2 induced bone regeneration by SDF-1α mediated stemcell recruitment,” Tissue Engineering Part A, vol. 20, no. 3-4,pp. 810–818, 2014.

[36] I. Majore, P. Moretti, F. Stahl, R. Hass, and C. Kasper, “Growthand differentiation properties of mesenchymal stromal cellpopulations derived from whole human umbilical cord,” StemCell Reviews and Reports, vol. 7, no. 1, pp. 17–31, 2011.

[37] S. Karahuseyinoglu, O. Cinar, E. Kilic et al., “Biology of stemcells in human umbilical cord stroma: in situ and in vitro sur-veys,” Stem Cells, vol. 25, no. 2, pp. 319–331, 2007.

[38] D. J. Hadjidakis and I. I. Androulakis, Bone Remodeling,Annals of the New York Academy of Sciences, 2006.

[39] U. Kini and B. N. Nandeesh, “Physiology of bone formation,remodeling, and metabolism,” in Radionuclide and HybridBone Imaging, pp. 29–57, Springer, Berlin, Heidelberg, 2012.

[40] L. J. Raggatt and N. C. Partridge, “Cellular and molecularmechanisms of bone remodeling,” Journal of Biological Chem-istry, vol. 285, no. 33, pp. 25103–25108, 2010.

[41] S. Kohli and V. Kohli, “Role of RANKL-RANK/osteoproteg-erin molecular complex in bone remodeling and its immuno-pathologic implications,” Indian Journal of Endocrinologyand Metabolism, vol. 15, no. 3, pp. 175–181, 2011.

[42] T. L. Andersen, M. E. Abdelgawad, H. B. Kristensen et al.,“Understanding coupling between bone resorption and forma-tion,” The American Journal of Pathology, vol. 183, no. 1,pp. 235–246, 2013.

[43] M. Borhani-Haghighi, T. Talaei-Khozani, M. Ayatollahi, andZ. Vojdani, “Wharton’s jelly-derived mesenchymal stem cellscan differentiate into hepatocyte-like cells by HepG2 cell lineextract,” Iranian Journal of Medical Sciences, vol. 40, no. 2,pp. 143–151, 2015.

[44] D. Bharti, S. B. Shivakumar, J.-K. Park et al., “Comparativeanalysis of human Wharton’s jelly mesenchymal stem cellsderived from different parts of the same umbilical cord,” Celland Tissue Research, vol. 372, no. 1, pp. 51–65, 2018.

[45] D. T. Denhardt and M. Noda, “Osteopontin expression andfunction: role in bone remodeling,” Journal of Cellular Bio-chemistry, vol. 72, no. S30-S31, pp. 92–102, 1998.

[46] J. Klein-Nulend, J. Roelofsen, C. M. Semeins, A. L. J. J. Bronck-ers, and E. H. Burger, “Mechanical stimulation of osteopontinmRNA expression and synthesis in bone cell cultures,” Journalof Cellular Physiology, vol. 170, no. 2, pp. 174–181, 1997.

[47] T. M. Liu and E. H. Lee, “Transcriptional regulatory cascadesin Runx2-dependent bone development,” Tissue EngineeringPart B: Reviews, vol. 19, no. 3, pp. 254–263, 2013.

[48] T. Komori, “Signaling networks in RUNX2-dependent bonedevelopment,” Journal of Cellular Biochemistry, vol. 112,no. 3, pp. 750–755, 2011.

[49] T. Komori, H. Yagi, S. Nomura et al., “Targeted disruption ofCbfa1 results in a complete lack of bone formation owing tomaturational arrest of osteoblasts,” Cell, vol. 89, no. 5,pp. 755–764, 1997.

[50] Q. Tu, J. Zhang, L. James et al., “Cbfa1/Runx2-deficiencydelays bone wound healing and locally delivered Cbfa1/Runx2

promotes bone repair in animal models,” Wound Repair andRegeneration, vol. 15, no. 3, pp. 404–412, 2007.

[51] J. H. Jonason, G. Xiao, M. Zhang, L. Xing, and D. Chen, “Post-translational regulation of Runx2 in bone and cartilage,” Jour-nal of Dental Research, vol. 88, no. 8, pp. 693–703, 2009.

[52] R. T. Franceschi, G. Xiao, D. Jiang, R. Gopalakrishnan, S. Yang,and E. Reith, “Multiple signaling pathways converge on theCbfa1/Runx2 transcription factor to regulate osteoblast differ-entiation,” Connective Tissue Research, vol. 44, no. 1, pp. 109–116, 2003.

[53] M. D. Yazid, S. H. Z. Ariffin, S. Senafi, M. A. Razak, and R. M.A. Wahab, “Determination of the differentiation capacities ofmurines’ primary mononucleated cells and MC3T3-E1 cells,”Cancer Cell International, vol. 10, no. 1, p. 42, 2010.

[54] F. Ng, S. Boucher, S. Koh et al., “PDGF, TGF-β, and FGF sig-naling is important for differentiation and growth of mesen-chymal stem cells (MSCs): transcriptional profiling canidentify markers and signaling pathways important in differ-entiation of MSCs into adipogenic, chondrogenic, and osteo-genic lineages,” Blood, vol. 112, no. 2, pp. 295–307, 2008.

[55] G. Xiao, D. Jiang, P. Thomas et al., “MAPK pathways activateand phosphorylate the osteoblast-specific transcription factor,Cbfa1,” Journal of Biological Chemistry, vol. 275, no. 6,pp. 4453–4459, 2000.

[56] N. Rucci, “Molecular biology of bone remodelling,” ClinicalCases in Mineral and Bone Metabolism, vol. 5, no. 1, pp. 49–56, 2008.

[57] L. F. Bonewald and G. R. Mundy, “Role of transforminggrowth factor-beta in bone remodeling,” Clinical Orthopaedicsand Related Research, no. 250, pp. 261–276, 1990.

[58] J. M. Hock, M. Centrella, and E. Canalis, “Insulin-like growthfactor I has independent effects on bone matrix formation andcell replication,” Endocrinology, vol. 122, no. 1, pp. 254–260,1988.

[59] R. M. Locklin, R. O. Oreffo, and J. T. Triffitt, “Effects of TGFβand BFGF on the differentiation of human bone marrow stro-mal fibroblasts,” Cell Biology International, vol. 23, no. 3,pp. 185–194, 1999.

[60] E. Torreggiani, G. Lisignoli, C. Manferdini et al., “Role of slugtranscription factor in human mesenchymal stem cells,” Jour-nal of Cellular and Molecular Medicine, vol. 16, no. 4, pp. 740–751, 2012.

[61] Á. Szepesi, Z. Matula, A. Szigeti et al., “In vitro characterizationof human mesenchymal stem cells isolated from different tis-sues with a potential to promote complex bone regeneration,”Stem Cells International, vol. 2016, Article ID 3595941, 9pages, 2016.

[62] A. Subramanian, C. Y. Fong, A. Biswas, and A. Bongso, “Com-parative characterization of cells from the various compart-ments of the human umbilical cord shows that theWharton’s jelly compartment provides the best source of clin-ically utilizable mesenchymal stem cells,” PLoS One, vol. 10,no. 6, article e0127992, 2015.

[63] F. Bustos, H. Sepúlveda, C. P. Prieto et al., “Runt-relatedtranscription factor 2 induction during differentiation ofWharton’s jelly mesenchymal stem cells to osteoblasts isregulated by Jumonji AT-rich interactive domain 1B histonedemethylase,” Stem Cells, vol. 35, no. 12, pp. 2430–2441,2017.

[64] Z. Qu, S. Mi, and G. Fang, “Clinical study on treatment of bonenonunion with MSCs derived from human umbilical cord,”


Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi, vol. 23, no. 3,pp. 345–347, 2009.

[65] P. Li, K. Cui, B. Zhang et al., “Transplantation of humanumbilical cord-derived mesenchymal stems cells for the treat-ment of Becker muscular dystrophy in affected pedigree mem-bers,” International Journal of Molecular Medicine, vol. 35,no. 4, pp. 1051–1057, 2015.

[66] I. H. Dilogo, M. R. A. Primaputra, J. A. Pawitan, and I. K. Liem,“Modified Masquelet technique using allogeneic umbilicalcord-derived mesenchymal stem cells for infected non-unionfemoral shaft fracture with a 12 cm bone defect: a case report,”International Journal of Surgery Case Reports, vol. 34, pp. 11–16, 2017.

[67] J. Cai, Z. Wu, L. Huang et al., “Cotransplantation of bone mar-row mononuclear cells and umbilical cord mesenchymal stemcells in avascular necrosis of the femoral head,” Transplanta-tion Proceedings, vol. 46, no. 1, pp. 151–155, 2014.

[68] M. Shi, S. Yu, R. Xu, and F. S. Wang, “Human mesenchymalstem cell therapy is feasible for liver cirrhosis associated withHBV infection,” Hepatology International, vol. 10, no. 1,pp. S341–S342, 2016.

[69] N. Morishima, K. Nakanishi, K. Tsuchiya, T. Shibata, andE. Seiwa, “Translocation of Bim to the endoplasmic reticulum(ER) mediates ER stress signaling for activation of caspase-12during ER stress-induced apoptosis,” Journal of BiologicalChemistry, vol. 279, no. 48, pp. 50375–50381, 2004.

[70] A. Patel and N. Riordan, “Allogeneic transplantation of humanumbilical cord mesenchymal stem cells (UC-MSC) for a singlemale patient with duchenne muscular dystrophy (DMD),”Cytotherapy, vol. 17, no. 6, article S46, 2015.

[71] D.-W. Kim, M. Staples, K. Shinozuka, P. Pantcheva, S.-D. Kang, and C. Borlongan, “Wharton’s jelly-derived mesen-chymal stem cells: phenotypic characterization and optimizingtheir therapeutic potential for clinical applications,” Interna-tional Journal of Molecular Sciences, vol. 14, no. 6,pp. 11692–11712, 2013.

[72] A. Can, F. T. Celikkan, and O. Cinar, “Umbilical cord mesen-chymal stromal cell transplantations: a systemic analysis ofclinical trials,” Cytotherapy, vol. 19, no. 12, pp. 1351–1382,2017.

[73] M. S. Choudhery, M. Badowski, A. Muise, J. Pierce, and D. T.Harris, “Donor age negatively impacts adipose tissue-derivedmesenchymal stem cell expansion and differentiation,” Journalof Translational Medicine, vol. 12, no. 1, p. 8, 2014.

[74] O. Karadas, D. Yucel, H. Kenar, G. Torun Kose, and V. Hasirci,“Collagen scaffolds with in situ-grown calcium phosphate forosteogenic differentiation of Wharton’s jelly and menstrualblood stem cells,” Journal of Tissue Engineering and Regenera-tive Medicine, vol. 8, no. 7, pp. 534–545, 2014.

[75] M. Torkaman, M. Ghollasi, M. Mohammadnia-Afrouzi,A. Salimi, and A. Amari, “The effect of transplanted humanWharton’s jelly mesenchymal stem cells treated with IFN-γon experimental autoimmune encephalomyelitis mice,” Cellu-lar Immunology, vol. 311, pp. 1–12, 2017.

[76] K. O’Donoghue, M. Choolani, J. Chan et al., “Identification offetal mesenchymal stem cells in maternal blood: implicationsfor non-invasive prenatal diagnosis,” Molecular HumanReproduction, vol. 9, no. 8, pp. 497–502, 2003.

[77] D. Baksh, R. Yao, and R. S. Tuan, “Comparison of proliferativeand multilineage differentiation potential of human mesen-chymal stem cells derived from umbilical cord and bone mar-row,” Stem Cells, vol. 25, no. 6, pp. 1384–1392, 2007.

[78] I. Arutyunyan, A. Elchaninov, A. Makarov, andT. Fatkhudinov, “Umbilical cord as prospective source formesenchymal stem cell-based therapy,” Stem Cells Interna-tional, vol. 2016, Article ID 6901286, 17 pages, 2016.

[79] J. Lim, Z. R. M. Razi, J. Law, A. M. Nawi, R. B. H. Idrus, andM. H. Ng, “MSCs can be differentially isolated from maternal,middle and fetal segments of the human umbilical cord,”Cytotherapy, vol. 18, no. 12, pp. 1493–1502, 2016.

[80] S. Kargozar, M. Mozafari, S. J. Hashemian et al., “Osteogenicpotential of stem cells-seeded bioactive nanocomposite scaf-folds: a comparative study between human mesenchymal stemcells derived from bone, umbilical cord Wharton’s jelly, andadipose tissue,” Journal of Biomedical Materials Research PartB: Applied Biomaterials, vol. 106, no. 1, pp. 61–72, 2018.

[81] M. R. Todeschi, R. el Backly, C. Capelli et al., “Transplantedumbilical cord mesenchymal stem cells modify the in vivomicroenvironment enhancing angiogenesis and leading tobone regeneration,” Stem Cells and Development, vol. 24,no. 13, pp. 1570–1581, 2015.

[82] L. Polo-Corrales, M. Latorre-Esteves, and J. E. Ramirez-Vick,“Scaffold design for bone regeneration,” Journal ofNanoscience and Nanotechnology, vol. 14, no. 1, pp. 15–56,2014.

[83] A. K. Batsali, C. Pontikoglou, D. Koutroulakis et al., “Differen-tial expression of cell cycle and WNT pathway-related genesaccounts for differences in the growth and differentiationpotential of Wharton’s jelly and bone marrow-derived mesen-chymal stem cells,” Stem Cell Research & Therapy, vol. 8, no. 1,p. 102, 2017.

[84] A. Zajdel, M. Kałucka, E. Kokoszka-Mikołaj, and A. Wilczok,“Osteogenic differentiation of human mesenchymal stem cellsfrom adipose tissue andWharton’s jelly of the umbilical cord,”Acta Biochimica Polonica, vol. 64, no. 2, pp. 365–369, 2017.

[85] R. K. Schneider, A. Puellen, R. Kramann et al., “The osteogenicdifferentiation of adult bone marrow and perinatal umbilicalmesenchymal stem cells and matrix remodelling in three-dimensional collagen scaffolds,” Biomaterials, vol. 31, no. 3,pp. 467–480, 2010.

[86] S. Bose, M. Roy, and A. Bandyopadhyay, “Recent advances inbone tissue engineering scaffolds,” Trends in Biotechnology,vol. 30, no. 10, pp. 546–554, 2012.

[87] H. W. Sun, R. J. Feigal, and H. H. Messer, “Cytotoxicity of glu-taraldehyde and formaldehyde in relation to time of exposureand concentration,” Pediatric Dentistry, vol. 12, no. 5, pp. 303–307, 1990.

[88] S. Mechiche Alami, H. Rammal, C. Boulagnon-Rombi et al.,“Harnessing Wharton’s jelly stem cell differentiation intobone-like nodule on calcium phosphate substrate withoutosteoinductive factors,” Acta Biomaterialia, vol. 49, pp. 575–589, 2017.

[89] L. Penolazzi, S. Mazzitelli, R. Vecchiatini et al., “Human mes-enchymal stem cells seeded on extracellular matrix-scaffold:viability and osteogenic potential,” Journal of Cellular Physiol-ogy, vol. 227, no. 2, pp. 857–866, 2012.

[90] K. E. Mitchell, M. L. Weiss, B. M. Mitchell et al., “Matrix cellsfrom Wharton’s jelly form neurons and glia,” Stem Cells,vol. 21, no. 1, pp. 50–60, 2003.

[91] Y. S. Fu, C. H. Lu, K. A. Chu et al., “Xenograft of human umbil-ical mesenchymal stem cells from Wharton’s jelly differentiat-ing into osteocytes and reducing osteoclast activity reversesosteoporosis in ovariectomized rats,” Cell Transplantation,vol. 27, no. 1, pp. 194–208, 2018.


[92] F. E. Al Jofi, T. Ma, D. Guo et al., “Functional organic cationtransporters mediate osteogenic response to metformin inhuman umbilical cord mesenchymal stromal cells,” Cytother-apy, vol. 20, no. 5, pp. 650–659, 2018.

[93] A. Ramesh, M.M. Kanafi, and R. R. Bhonde, “Modulus-depen-dent characteristics of Wharton’s jelly mesenchymal stem cells(WJMSC) encapsulated in hydrogel microspheres,” Journal ofBiomaterials Science, Polymer Edition, vol. 25, no. 17,pp. 1946–1961, 2014.

[94] L. Wang, L. Zhao, and M. S. Detamore, “Human umbilicalcord mesenchymal stromal cells in a sandwich approach forosteochondral tissue engineering,” Journal of Tissue Engineer-ing and Regenerative Medicine, vol. 5, no. 9, pp. 712–721, 2011.


Hindawiwww.hindawi.com

International Journal of

Volume 2018

Zoology

Hindawiwww.hindawi.com Volume 2018

Anatomy Research International

PeptidesInternational Journal of



Journal of Parasitology Research

GenomicsInternational Journal of


Hindawi Publishing Corporation http://www.hindawi.com Volume 2013Hindawiwww.hindawi.com

The Scientific World Journal

Volume 2018


BioinformaticsAdvances in

Marine BiologyJournal of



Neuroscience Journal


BioMed Research International

Cell BiologyInternational Journal of



Biochemistry Research International

ArchaeaHindawiwww.hindawi.com Volume 2018


Genetics Research International


Advances in

Virolog y Stem Cells International



Enzyme Research


International Journal of

MicrobiologyHindawiwww.hindawi.com

Nucleic AcidsJournal of

Volume 2018

Submit your manuscripts atwww.hindawi.com

https://www.hindawi.com/journals/ijz/

https://www.hindawi.com/journals/ari/

https://www.hindawi.com/journals/ijpep/

https://www.hindawi.com/journals/jpr/

https://www.hindawi.com/journals/ijg/

https://www.hindawi.com/journals/tswj/

https://www.hindawi.com/journals/abi/

https://www.hindawi.com/journals/jmb/

https://www.hindawi.com/journals/neuroscience/

https://www.hindawi.com/journals/bmri/

https://www.hindawi.com/journals/ijcb/

https://www.hindawi.com/journals/bri/

https://www.hindawi.com/journals/archaea/

https://www.hindawi.com/journals/gri/

https://www.hindawi.com/journals/av/

https://www.hindawi.com/journals/sci/

https://www.hindawi.com/journals/er/

https://www.hindawi.com/journals/ijmicro/

https://www.hindawi.com/journals/jna/

https://www.hindawi.com/

https://www.hindawi.com/

Documents

Osteogenic Induction of Wharton s Jelly-Derived ...downloads.hindawi.com/journals/sci/2018/2406462.pdf · Review Article Osteogenic Induction of Wharton’s Jelly-Derived Mesenchymal