The Egyptian Rheumatologist (2014) 36, 179–186

HO ST E D BYEgyptian Society of Rheumatic Diseases

The Egyptian Rheumatologist

www.rheumatology.eg.netwww.elsevier.com/locate/ejr

ORIGINAL ARTICLE

Effect of intra-articular injection of mesenchymal

stem cells in cartilage repair in experimental

animals

* Corresponding author. Tel.: +20 226223833.

E-mail address: irene_raouf@yahoo.com (I.R. Amin).

Peer review under responsibility of Egyptian Society of Rheumatic

Diseases.

http://dx.doi.org/10.1016/j.ejr.2014.03.001

1110-1164 � 2014 Production and hosting by Elsevier B.V. on behalf of Egyptian Society of Rheumatic Diseases.

Open access under CC BY-NC-ND license.

Open access under CC BY-NC-ND license.

Nadia Salah Kamela, Mona Mahmoud Arafa

a, Amr Nadim

d,

Hnaa Amer b, Irene Raouf Amin a,*, Naglaa Samir c, Amina Salem a

a Department of Physical Medicine, Rheumatology and Rehabilitation, Faculty of Medicine, Ain Shams University, Egyptb Department of Clinical Pathology, Faculty of Medicine, Ain Shams University, Egyptc Department of Pathology, Faculty of Medicine, Ain Shams University, Egyptd Department of Obstetrics and Gynecology, Faculty of Medicine, Ain Shams University, Egypt

Received 10 February 2014; accepted 8 March 2014Available online 18 April 2014

KEYWORDS

Intra articular injection;

Mesenchymal stem cells

(MSCs);

Cartilage repair;

Osteoarthritis;

Experimental animals

Abstract Osteoarthritis (OA) is characterized by degeneration of the articular cartilage and,

ultimately, joint destruction. Human umbilical cord blood (hUCB) may prove to be a new source

of mesenchymal stem cells (MSCs) for cellular therapeutics used for cartilage repair.

Aim of the work: This study was carried out over a nine-month period of time, to study the effect

of intra-articular injection of hUCB MSCs in cartilage repair by histopathological and ultra

structural assessment.

Materials and methods: We conducted our study on 20 adult rats, which were subjected to the

induction of cartilaginous defect in both knee joints. This was followed by injection of MSCs

suspended in Hyaluronic acid solution in the right knee of each rat while the left knee served as

a control. Histopathological and electron microscopic studies were performed.

Results: The present study revealed: In the injected knees; In 73% of the cases, the tissue was

typical of fibrohyaline cartilage and appeared more cellular than fibrous. In 27% of the cases the

repaired tissue appeared more fibrous than hyaline. In the control knees; the newly formed tissue

was an undifferentiated connective tissue and the cells were covered with a thin layer of fibrous

tissue. The electron microscopic pictures of the injected knees showed mitotic chondrocyte activity.

The pictures indicated a repaired fibrohyaline cartilage.

Conclusion: We can conclude that the intra-articular injection of hUCB MSCs is an effective

method for cartilage repair in rats. This makes it a very promising tool for the treatment of patients

with OA.� 2014 Production and hosting by Elsevier B.V. on behalf of Egyptian Society of Rheumatic Diseases.

180 N.S. Kamel et al.

1. Introduction

Osteoarthritis (OA) is a degenerative joint disease that is char-acterized by erosion of the articular cartilage, growth of bone

at the margins i.e., osteophytes, subchondral sclerosis and arange of biochemical and morphologic alterations of the syno-vial membrane and joint capsule [1]. The primary tissue

affected is the thin rim of hyaline articular cartilage interposedbetween the two articulating bones [2].

In early OA, the articular cartilage surface becomesirregular, and superficial clefts within the tissue become

apparent. As the condition worsens, the clefts deepen, surfaceirregularities increase and the articular cartilage eventuallyulcerates, exposing the underlying bone [3]. Chondrocytes in

areas surrounding an injured zone are unable to migrate,proliferate, repopulate, regenerate or repair tissue with similarstructure, function, and biomechanical properties of normal

hyaline cartilage [4].The burden of OA is exacerbated by the inadequacies of

current therapies. Nonpharmacologic and pharmacologic

treatments are used for early and moderately early cases ofOA [5].

Mesenchymal stem cells (MSCs), which have the ability todifferentiate into cells of the chondrogenic lineage, are very

promising candidates to develop new cell-based articular carti-lage repair strategies; this strategy entails the use of MSCs astrophic producers of bioactive factors to initiate endogenous

regenerative activities in the OA joint [6]. Autologous bonemarrow (BM) represents the main source of MSCs for bothexperimental and clinical studies; however as the number of

MSCs and their differentiation capacity decline with age, theirtherapeutic potential might be diminished as well [7]. As sev-eral ethical and practical issues arise from the use of BM

and fetal stem cells, umbilical cord blood (UCB) has turnedout to be an excellent alternative source of MSCs for clini-cal-scale allogeneic transplantation [8].

It was postulated that hyaluronic acid might facilitate the

migration and adherence of MSCs to the defect, which mightexplain the occurrence of partial healing at 6 weeks in animalsthat were treated with hyaluronic acid alone. The repaired

tissue in animals treated with hyaluronic acid alone was ofinferior quality and was shown to deteriorate further after12 weeks, so the combination between MSCs and hyaluronic

acid will result in synergistic effect [9].Aim of the Work: This study was carried out over a nine-

month period of time, to study the effect of intra-articularinjection of human umbilical cord blood (hUCB) mesenchy-

mal stem cells (MSCs) in cartilage repair by histopathologicaland ultra structural assessment.

2. Materials and methods

The study was carried out on twenty albino rats of Wistarstrain (adult males) during a nine-month period of time. They

were brought from the medical research center, faculty ofmedicine, animal house of Ain Shams University. This studywas approved by the local ethical committee.

The animals were maintained under conditions ofcontrolled humidity, they were fed with commercial rat pelletsand water. The animal house staff detected the age and weight

of the animals and supervised their feeding. All animals werehealthy and had no joint problems.

2.1. All animals were subjected to the following

Induction of cartilaginous defect in both knee joints byscratching the cartilage using a sterile needle [10,11]. In

maximal flexion, longitudinal and diagonal grooves were madeon the weight-bearing parts of femoral condyles withoutdamaging the subchondral bone. The latter was checked by

histology in 2 of the rats which were sacrificed; one of themafter 1 week and the other after 4 weeks from the scratchingwhich was done in the beginning of the experiment in order

to ensure development of osteoarthritis (OA).

2.2. Preparation of MSCs from human umbilical cord blood(UCB)

2.2.1. Collection of UCB

Umbilical cord blood was obtained from the labor room of

Obstetrics and Gynecology Department, Faculty of Medicine,Ain Shams University after written consent from the mothers.

4 UCB samples from full-term deliveries were collected

from the unborn placenta using complete aseptic techniquein sterile 15 ml Falcon tubes (Nunclon, Germany) containing2 ml of acid citrate dextrose (ACD) anticoagulant (Lonza,Switzerland). The samples were stored at 22 ± 4 �C before

processing [12]. Isolation and culture of MSCs were carriedout in the medical research center, Faculty of Medicine, AinShams University as follows.

2.2.2. Isolation of mononuclear cells (MNCs)

Under complete asepsis each UCB sample was diluted 1:1 withphosphate-buffered saline (PBS) (lonza) and was carefully

loaded onto Ficoll–Hypaque solution 2:1 ratio. After densitygradient centrifugation at 2000 rpm for 30 min at roomtemperature, MNCs were removed from the interphase (the

Buffy coat) and were washed three times with PBS; each timewere centrifuged at 1500 rpm for 5 min, a clear cell pallet wasformed in the bottom of the tube [13].

2.2.3. Culture of mesenchymal stem cells

The cells were cultured in complete culture medium;Dulbecco’s modified Eagle’s medium (DMEM) (Lonza, Swit-

zerland) containing 12% fetal calf serum, 1% antibiotics –antimycotic; 100 units/ml of penicillin, 100 lg/ml ofstreptomycin and 250 lg/ml Amphotericin B. Cells were

placed in 25 ml Falkon flasks (Nunclon, Germany). The flaskswere incubated at 37 �C in 5% CO2 (NuAire, USA).

On the fifth day of culture, non-adherent cells werediscarded and adherent cells were examined microscopically

for morphological evaluation of spindle shaped cells. The com-plete medium was changed and the flasks were reincubated.Flasks were examined every other day for MSCs (spindle

shaped, fibroblast like cells).After 2 weeks of culture, the adherent cells were almost

confluent. Adherent cells were harvested using 0.25% trypsin

for 5 min at 37 �C, 5 ml of medium was added to deactivateit. Cells were then counted with a haemocytometer. Cells werethen collected in a 15 ml Falcon tube and centrifuged at

Effect of intra-articular injection of mesenchymal stem cells in cartilage repair in experimental animals 181

2000 rpm for 10 min. The supernatant discarded and thesediment washed twice with PBS, and then centrifuged1500 rpm for 5 min [14].

2.2.4. Preparing MSCs for injection

After isolating MSCs we suspended them in 2 ml ofHyaluronic acid solution (Hyalgan�, Sanofi Aventis, USA)

at a density of 1.2–1.5 · 106 cells/ml.

2.3. Injection of animals

Injection of MSCs suspended in Hyaluronic acid solution wasdone in the right knee of each rat after sterilization withBetadine solution. The left knee served as a control [15].

2.4. Preparation of histological sections of the knee joint

Animals were sacrificed by intraperitoneal injection of a lethal

dose of thiopental (50 mg/kg) after 6 weeks of injection andknee joints were removed. Histopathological study was doneto examine chondrogenic regenerative changes of the animal

articular cartilage after injection [16].The total knee joints were removed and fixed for 4 days in

10% neutral formalin, decalcified by 5% formic acid indistilled water. The decalcifying solution was renewed every

48 h until softening of the tissues. The decalcified specimenswere washed and dehydrated in ascending grades of alcohol,cleared in xylene and embedded in paraffin. From each

paraffin block, longitudinal sections (5 lm) were cut andstained with hematoxylin & eosin and Masson’s trichrome(for detection of collagen fibers).

2.5. Experimental assessment

Our experimental assessment was done to detect the develop-ment of OA in the knees of the studied rats and this depended

upon subjective observational parameters as limping and/oraggressive behavior denoting pain.

2.6. Histopathological assessment

Each stained section was examined with a light microscope toassess the following criteria for cartilage repair: (1) Tissue

morphology (2) Surface architecture (3) Clustering of chon-drocytes (4) Thickening of repaired cartilage (5) Synovial cellproliferation and inflammation.

The control slides were examined to assess the followingcriteria for osteoarthritic changes: (1) Surface irregularities(2) Organization and hypertrophy of chondrocytes and (3)The degree of proliferation and inflammation of synovial cells.

2.7. Preparation for electron microscopic (EM) study

The right (injected) knee joint of one of the knees was opened

anteriorly by cutting through the ligamentum patellae. Thearticular cartilage surface was immediately washed with saline.Washing continued while the knee was disarticulated. The soft

tissue surrounding the femur was reflected proximally and the

limb amputated at the middle of femoral shaft. Specimen thusconsisted of the distal half of the femur.

2.8. EM preparation of the sample

First, the specimen was immersed in freshly prepared glutaral-dehyde, buffered with 0.1 M sodium cocodylate at pH 7.3.The

specimen was then left for 4 h at a temperature of four degreescentigrade. The specimen was then washed for 2 h in the threechanges of the same buffer. Then, it was post-fixed in 1%

osmium tetroxide for two hours in the refrigerator. The speci-men was then washed in three changes of the same buffer forhalf an hour in each change. After post-fixation, dehydration

of the specimen was carried out through ascending grades ofethanol alcohol at four degrees centigrade as follows: 30 minin each of the following concentrations of ethanol alcohol,50%, 70%, 80% and 95%. Then, we used three changes alco-

hol for 30 min. At room temperature, the specimen was treatedwith three changes of propylene oxide over a period of one anda half hours. The specimen was impregnated in a mixture of

equal parts of epoxy resin mixture and propylene oxide for8 h. Finally, specimen was embedded in pure resin and was leftfor 48 h in an oven at 60 �C for polymerization of the resin.

Section cutting was done on an LKB ultramicrotome. Ultrathin sections were obtained and picked up on coated coppergrids. The ultrathin sections were stained with urenyl acetatefor 30 min, and then followed by lead citrate for 15 min. The

stained ultrathin sections were examined under transmissionelectron microscope; Sumy electron optics (TEM-100 SEO)Ukraine, at 60 kV accelerating voltage.

Electron microscopic study was done to visualize mitoticpictures (newly formed chondrocytes and chondroblasts), alsoto assess the collagen fibers and the matrix arrangement.

Statistical analysis: Data collected were presented in theform of numbers and percentages.

3. Results

This study was carried out on twenty albino rats of Wistarstrain (adult males). Their age ranged from 6 to 12 months

and their weight ranged from 180 to 220 g. All the animals weresubjected to induction of cartilaginous defect in both kneejoints by scratching the cartilage using a sterile needle in orderto develop OA. Experimental assessment, in the form of obser-

vation of limping and/or aggressive behavior denoting pain,revealed development of OA in all the studied rats. The 2 ratswhich were sacrificed after 1 and 4 weeks from the scratching

maneuver showed evidence of development of OA in the formof fibrillation of the surface, the chondrocytes were smaller andlying in parallel longitudinal rows, the synovial cells showed

mild proliferation and mild infiltration by inflammatory cells.After induction of OA by scratching, one of the animals

died before being injected. After 6 weeks of scratching; injec-

tion of MSCs suspended in hyaluronic acid solution was donein the right knee of 17 rats and the left knee served as a control.After injection none of the animals was limping or aggressive.

The animals were killed after 6 weeks of injection and knee

joints were removed. Two of the animal samples were used inthe electron microscopic study and the rest of the 15 animalsamples were used for the histopathological assessment.

Figure 1 (a) Control knees: Granulation tissue at the site of

articular injury (H&E ·400).

182 N.S. Kamel et al.

3.1. Microscopic findings

In the control group (left knees) (Table 1): the area of thedefect was partially filled with a fibrovascular granulationtissue, which united the wound edges (Fig. 1a). The newly

formed tissue was composed of undifferentiated connectivetissue cells covered with a thin layer of fibrous tissue. It wasgrossly distinguishable from the surrounding normal tissue.Cartilaginous sequestra surrounded by organized fibrous tissue

were seen (Fig. 1b). All the control knees did not show anysigns of cartilage repair.

In the injected group (right knees) (Table 2): no signs of OA

such as osteophytes, cysts formation, cartilage erosion or syno-vial proliferation were observed in any of the knees; fibrocar-tilaginous mass was detected at the region of the defect by

Masson’s trichrome stain in 4 treated knees (27%). The tissueappeared more fibrous than hyaline. The surface became mod-erately fibrillated, the chondrocytes were smaller and lying in

parallel longitudinal rows, the subjacent matrix was denselyfibrous and the synovial cells showed mild proliferation andmild infiltration by inflammatory cells (Fig. 2). In theremaining 11 treated knees (73%): the tissue was typical of

fibro-hyaline cartilage and appeared more cellular thanfibrous. These findings were confirmed by Masson’s trichromestain that demonstrated pink nuclei subjacent to the pale blue

staining matrix and fine fibrous tissue network showing intensemetachromasia that divided the chondrocytes into clusters(Figs. 3a–c).

3.2. Electron microscopic findings

As regards the EM study of the MSC-injected knees; it showeda repaired fibro-hyaline cartilage demonstrated by many

chondrocytes and chondroblasts. The chondrocytes hadcentrally positioned nuclei surrounded by a rich roughendoplasmic reticulum (rER) and numerous mitochondria.

The chondrocytes also showed chondrocyte mitotic activity.The matrix displayed fine collagen fibrils (Fig. 4a). In someareas thick bundles of collagen fibrils, which were sectioned

Table 1 Histopathological findings of the control knees (Lt. side).

No Joint surface Chondrocytes

Irregularity Disorganization

1 2 1

2 1 1

3 2 2

4 1 1

5 2 1

6 1 1

7 2 2

8 1 2

9 1 1

10 1 1

11 1 1

12 2 2

13 1 1

14 2 2

15 2 2

Normal = 0, Mild = 1, Moderate = 2, Severe = 3.

both transversely and longitudinally, were seen. Individualfibrils displayed alternating transverse dark and light bandingalong their length (Fig. 4b). The pictures indicated a repaired

fibro-hyaline cartilage.

4. Discussion

Osteoarthritis is a major cause of disability in the elderly; theprevalence of this disease is expected to increase dramaticallyover the next 20 years with aged population [17,18]. The

burden of OA is exacerbated by the inadequacies of currenttherapies. Non-pharmacologic and pharmacologic treatmentsare used for early and moderately early cases of OA, but pro-

tection of articular cartilage has so far not been convincingly

Synovial cells

Hypertrophy Proliferation Inflammation

1 2 2

1 2 2

1 1 1

1 2 2

1 2 2

1 1 1

1 2 2

2 2 2

1 1 1

1 1 1

1 1 1

1 2 2

1 1 1

2 2 2

2 2 2

Figure 1 (b) Control knees: Organization and formation of

cartilaginous sequestra surrounded by disorganized fibrous tissue

(H&E ·200).

Figure 2 Injected Knee: Chondrofication of old granulation

tissue (arrow) with fibrillated surface (star) (H&E ·200).

Effect of intra-articular injection of mesenchymal stem cells in cartilage repair in experimental animals 183

shown [5,19]. Surgical intervention is often indicated when thesymptoms cannot be controlled and the disease progresses [20].Whether arthroscopic lavage and/or debridement can providesymptomatic relief is unclear [21]. Consequently, patients with

severe OA are currently excluded from these treatments [22].Rats as animal models have a short life span, are available,

cheap and well adapted to our climate, these characters made

them ideal for this study. Animals used in this study were adultrats because the cartilage of adult animals is resistant to repairthan in immature animals and therefore more similar to adult

human subjects [23]. We excluded females because they usuallyget pregnant which may disturb the study by additional factorsout of the scope of our study.

Scratching the cartilage has the advantages that it is easy toperform and it induces partial thickness cartilage defect thatdoes not reach the subchondral bone, so there is no involve-ment of the vasculature. Consequently, progenitor cells in

blood and marrow cannot enter the damaged region to

Table 2 Histopathological findings of the mesenchymal stem cells (

No Tissue

morphology

Surface architecture

(irregularity)

Chondrocyte

clustering

C

th

1 HF 2 2 2

2 F 1 2 1

3 HF 2 2 2

4 HF 2 2 2

5 HF 2 2 2

6 HF 2 2 2

7 F 1 2 1

8 HF 2 2 2

9 F 1 2 1

10 HF 1 2 1

11 HF 1 2 1

12 HF 1 2 1

13 F 1 2 1

14 HF 2 2 1

15 HF 2 2 2

Normal = 0, Mild = 1, Moderate = 2, Severe = 3. Fibro cartilage = F,

influence or contribute to the reparative process to ensure that

any chondral repair is entirely from the injected cells [10].Since most OA models use knee joints, an important con-

sideration in the use of these models is the load-bearing patternof the species being used [24]. So we did not prevent the animal

from weight bearing after induction of the cartilage defect, toeliminate the rest factor that may share in the healing processand we started treatment after 6 weeks of scratching to ensure

that the OA is well established. On the other hand, Yanai et al.[25] established an animal model of large full-thicknessarticular cartilage defects in the knee of rabbits using a hinged

fixator and considered joint distraction to be favorable for theregeneration.

Human umbilical cord blood (hUCB) MSCs have many

advantages because of the immaturity of newborn cells com-pared with adult cells also could be more convenient for celltransplantation and be a more economical source of MSCs,than bone marrow MSC. Furthermore, hUCB provides no

ethical problems for basic studies and clinical applications[26]. The study of Erices et al. [27] provided strong evidence

MSCs) injected knees (Rt. Side).

artilage

ickening

Synovial cells Dose of injected

cells (million cell)Proliferation Inflammation

1 1 1

2 2 1

1 1 1

1 1 1

1 1 1

1 1 1

1 1 5

1 1 5

1 1 5

1 1 5

1 1 3

1 1 3

2 2 3

1 1 3

1 1 3

Hyaline-Fibrocartilage = HF.

Figure 3 (c) Injected Knee: Hypercellular hyaline-like cartilage

in which the fibrous network divides the chondrocytes into clusters

(Masson’s trichrom ·400).

Figure 3 (b) Injected Knee: Mature chondrocyte with subjacent

fibrohyaline matrix (H&E ·400).

Figure 4 (a) Chondrocytes surrounded by fine collagen fibrils

(TEM ·3000).

Figure 3 (a) Injected Knee: Fibrohyaline cartilage covering the

articular surface (H&E ·200).

Figure 4 (b) Bundles of collagen fibrils (TEM ·15,000).

184 N.S. Kamel et al.

for the presence of circulating non-hematopoietic stem cells inhUCB. Rosada et al. [28] has reported that cord blood

multilineage cells are slower to establish in culture, have alower precursor frequency and a lower level of bone antigenexpression, and lack constitutive expression of neural antigens

when compared with bone marrow, suggesting a more primi-tive population. The hUCB is easy to obtain and to store,and its use for hematopoietic stem cell transplantation does

not require a close human leucocyte antigen match as reportedby Gluckman [29].

Human UCB stem cells are xenograft cells. Rat UCB is dif-

ficult to obtain and the available quantity of rat UCB is verylimited; however, hUCB is easy to obtain and is plentiful. Inour experiment, we expected to evaluate only the possibilityof UCB stem cells differentiated into chondrocytes in vivo.

So, we used hUCB in the rat model. Umbilical cord bloodmay prove to be a new source of cells for cellular therapeuticsfor stromal, bone, and potentially, cartilage repair. Also it is a

very promising source in Egypt because of the high birth rate.For all the above-mentioned reasons, cord blood MSCs were

Effect of intra-articular injection of mesenchymal stem cells in cartilage repair in experimental animals 185

considered in our study as the most promising cell source forcartilage repair.

Due to the specific structure and functions of placenta,

human extra-embryonic MSCs, including MSCs derived fromthe UCB represent stem cell types, which combine someproperties of pluripotent embryonic stem cells with other prop-

erties of multipotent MSCs. They have immunoprivilegedcharacteristics, posses a broader plasticity, and proliferatefaster than adult MSCs [30].

The hUCB MSCs were delivered in this study by intra-articular injection, there are 2 different approaches to this.One is to implant cells directly with or without a suitablematrix or scaffold seeded with chondroprogenitor cells and sig-

naling substances as in the study of Jackson and Simon [31].The alternative is to differentiate stem cells in vitro andimplant a mature construct. The ability of stem cells to differ-

entiate and adhere to scaffolds such as matrices of hyaluronanderivatives and gelatin-based resorbable sponge matrices hasbeen investigated and proven by Solchaga et al. [32], and

Ponticello et al. [33] respectively.Most studies presume that scaffolds are required for the

regeneration of cartilage. It is ventured that loads and fluid

movements would simply prevent cells from thriving wherethey are needed. However Barry [34] has shown that MSCscan survive and thrive without a scaffold and injected stemcells have been recovered in viable form in a goat knee with

simulated arthritis.In the present study hUCB MSCs were delivered by intra-

articular injection in a suspension of hyaluronic acid (HA)

solution without the use of a solid biomatrix 6 weeks afterscratching the cartilage. Lee et al. [9] postulated that HA mightfacilitate the migration and adherence of MSCs to the defect.

Our choice of experimental animals to be rats, limited ourability to experimental assessment. It was limited to subjectiveobservational parameters such as limping or aggressive behav-

ior denoting pain, so we chose the histopathological assess-ment and ultrastructural study that were based uponhistological objectives. Samples were stained with hematoxylin& eosin and Masson’s trichrome.

In the present study, the repair of lesions was a variable mix-ture of fibrous tissue, fibrocartilage and hyaline-like cartilage.At 6 weeks at the control group; the area of the defect was filled

with a fibrovascular granulation tissue, the newly formed tissuewas an undifferentiated connective tissue covered with a thinlayer of fibrous tissue. Cartilaginous sequestra surrounded by

organized fibrous tissue were seen. All control knees did notshow any signs of repair (differentiated cartilage) denoted thatthere is no systemic parenteral trafficking through systemic cir-culation from the injected right knee to the other left knee.

These findings coincide with those reported by Yanai et al.[25], Bos et al. [35], and Lee et al. [9]. However in this studythe tissue appeared more fibrous than cartilaginous.

At the injected group; no signs of OA such as osteophytes,cysts formation, cartilage erosion or synovial proliferation,were observed in any of the knees these findings match with

that of Lee et al. [9].After 6 weeks of injection fibrocartilaginous mass was

detected in 27% of cases. The tissue appeared more fibrous

than cartilaginous and the surface layers and the cells weremore typically fibrocartilaginous than hyaline. The surfacebecame moderately fibrillated, the chondrocytes are smallerand lying in parallel longitudinal rows, the subjacent matrix

was densely fibrous and the synovial cells showed mild prolif-eration and mild infiltration by inflammatory cells. In 73% ofcases; the tissue appeared more cellular than fibrous. The thin

fibrillated surface and the cells were typical of fibro-hyalinecartilage; these findings match with those of Yanai et al. [25],Yan and Yu [36], and Lee et al. [9].

Regarding the EM study; it showed a repaired fibrohyalinecartilage demonstrated by many chondrocytes and chondro-blasts. The pictures indicated a repaired fibrohyaline cartilage.

A recent study conducted by Nam and his colleagues [37],showed a superior role of injecting autologous bone marrowderived mesenchymal stem cells (BM-MSCs) to hUCB MSCs.This study demonstrated hyaline-like cartilage regeneration in

the knees of goats receiving BM-MSCs after two full-thicknesschondral 5 mm diameter defects were created. They suggestedthat supplementing intra-articular injections of BM-MSCs fol-

lowing bone marrow stimulation (BMS) knee surgery providessuperior cartilage repair outcomes.

We can conclude from these results that the intra-articular

injection of hUCB MSCs is an effective method for cartilagerepair in rats. This makes it a very promising tool for the treat-ment of patients with OA.

Conflict of interest

None.

Acknowledgement

The authors would like to express their gratitude to AwatefMohamed (Veterinary Fellow of Biochemistry) and Larissa

Inge (Electron Microscopy) without whom this work wouldnot have been accomplished.

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