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Moshiri A, Oryan A, Meimandi-Parizi A. Role of stem cell therapy in orthopaedic tissue engineering and regenerative medicine: a comprehensive review of the literature from basic to clinical application. Hard Tissue 2013 May 20;2(4):31.

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1

Role of stem cell therapy in orthopaedic tissue engineering and regenerative medicine: a

comprehensive review of the literature from basic to clinical application

A Moshiri1, A Oryan2, A Meimandi-Parizi1

1: Division of Surgery and Radiology, Department of Clinical Sciences, School of Veterinary Medicine, Shiraz University,

Shiraz, Iran

2: Department of Pathology, School of Veterinary Medicine, Shiraz University, Shiraz, Iran

Corresponding author: Dr Ali Moshiri, DVM, DVSc (Veterinary Surgery). E-mail: dr.ali.moshiri@gmail.com, cellphone,

+989123409835

2

Abstract

Treatment of soft and hard connective tissues especially those associated with massive tissue loss is technically

demanding. Tendon and cartilage have low healing capability. Massive bone injures are often associated with non-

union or malunion and other complications. Treatment of tendon-bone junction is challenging and treatment of

osteoarthritis is more palliative than curative. Tissue engineering which composed of three elements including

scaffolds, healing promotive factors such as growth factors, and stem cells is an option. Recently stem cell based

therapy is much popular due to the encouraging reported results. This review introduced stem cells and discussed

their potential application and roles in tissue regenerative medicine. We have focused on the effectiveness of stem

cells based therapy on different tissue injuries, including tendon, tendon to bone junction, bone, cartilage and

osteoarthritis. In vitro to clinical evidences have been discussed in detail with the aiming to conclude whether stem

cell based therapy is a clinically accepted method. This review showed that despite of exploring several sources for

the stem cells, the adult mesenchymal stem cells are still the only reliable stem cells in tissue regenerative medicine.

In addition, despite of significant improvement in tissue engineering, it seems cell seeding is technically demanding

and direct injection of the stem cells is the only reliable method for cell delivery at injured site. Because of

controversial results and lack of well-designed clinical trial studies, this review also showed that despite of several

animal studies and application of stem cells on various tissue injuries, it is still soon to suggest stem cell therapy as

an effective method in restoring morphology and functionality of the injured tissues.

Keywords: stem cells, tissue engineering, tendon, bone, cartilage, regeneration, healing

3

Introduction Treatment of injured tendon, ligament, cartilage and bone often has faced with some difficulties1-3. Such tissues

tolerate different types of forces such as stress, torsion and bending, during healing which affect their healing and if

not properly managed, ideal healing would not be expected4-8. In addition such soft and hard connective tissues have

low healing capability because their circulation is often impaired due to the injury2. Some of the tissue injuries can

be directly repaired surgically9,10. For example simple tendon ruptures could be sutured and anastomosed11. Simple

fractures could be fixed with a variety of external and internal fixation techniques and devices. However, some of

the tissue injuries are engaged with significant tissue loss and could not be retrieved by routine surgical methods2. In

large massive tendon injuries the gap should be reconstructed by auto or allografts2. In large massive bone fractures

or other types of bone injuries such as osteosarcoma there is a need to resect diseased bone, and despite of intensive

fixation of the bony ends, large gap is produced. In such condition the gap area should be reconstructed by a

cancellous bone graft or other types of graft based on the experience and preference of the surgeon12-21.

Tissue transplantation has its own limitations and it is widely accepted that there is a need for alternative solutions2.

There are also some other orthopedic injuries that could not be solved with only grafting methods. For example, in

ACL reconstruction, although a torn ACL is replaced by auto or allograft, however, major concerns exist for the

ligament to bone healing22,23. Most of the failures occur at this site and this is a major problem24. Healing of cartilage

is also extremely slow and if osteoarthritis (OA) happen the condition becomes more complicated and there is no

well accepted method for retrieving such conditions25-29. Most of the gold standard methods are palliative, and if

they are effective; they can only reduce the rate of OA progression30-32. Therefore, there is a need to find a new way

with the hope that such injuries will heal with minimum complication and be functional after the healing period.

Tissue engineering is one of these new approaches2,33. In the last decade, several advancements have been achieved

and the tissue engineering technologies and tissue engineered based products have been developed and produced33.

The initial tests have been represented encouraging results however, the results are still primitive and several animal

and clinical studies are needed to prove such a new option2. Basically, tissue engineering can be divided into three

major categories. Scaffolds are the first category and have many roles. They can be implanted to act as a tissue

4

conductor and guide the healing tissue in a desired manner2,10 or behave as a vehicle for growth factors and stem

cells2,3,10. The second category is the healing promotive factors2,3,10 such as growth factors. Basic fibroblast growth

factor (bFGF), bone morphogenic protein 2,4,7 (BMP 2,4 & 7), vasculoendothelial growth factor (VEGF), tissue

growth factor B (TGFB), platelet derived growth factors (PDGF) are some examples1-3,9. These growth factors have

many roles in tissue regeneration and regulate different stages of wound healing3. Platelet rich plasma (PRP) is a

simple and inexpensive source for the growth factors and can be widely used in clinical practice3.

Glycosaminoglycans such as hyaluronic acid and chondroitin sulfate have important roles during tissue healing5,11,34-

36 and could be considered as other healing promotive factors2. Finally, the third category is the stem cells. Much

advancement has been achieved in this field of regenerative medicine and the primitive results are encouraging.

Stem cells have crucial roles in tissue regeneration because they can collaborate in matrix synthesis and result in

potential enhancement of the quality and integrity of the healing tissues2. An ideal tissue engineering approach

should combine all these three elements to produce an appropriate tissue engineered graft2. However most of the

studies have focused on the role of each category and did not combined them in a manner to be applicable in clinical

practice2.

In this review we have introduced stem cells, their roles in tissue healing and provided some evidences to show

whether they are effective in restoring structure and function of the injured tissues. Several in vitro and in vivo

studies have been reviewed by the authors and the potential advancement and limitations of stem cells have been

discussed to open a new area of research and make new suggestions for those who are engaged with tissue

engineering and regenerative medicine.

Stem cells

Recently much attention has been paid on stem cells37,38. Stem cells have the potential to divide and differentiate

into various specialized cell types and can self-renew to produce more stem cells39 (Fig. 1 and 2).

Following tissue injury the inflammatory cells migrate into the wound area2. These cells together with the injured

cells of the tissue and coagulated platelets, release and deliver growth factors, cytokines and proinflammatory

mediators which have major roles in attracting mesenchymal stem cells to the lesion2,3,5-8,11. The mesenchymal stem

5

cells migrate into the injured area and under stimulation of such mediators, proliferate and differentiate into the

specific tissue cells2. Gradually these proliferated cells produce large amounts of matrix mainly glycosaminoglycans

such as hyaluronic acid, chondroitin sulfate and dermatan sulfate at earlier stages of wound healing and later, the

collagen and elastic fibrils2. Some of the stem cells are differentiated into the endothelial cells and produce new

blood vessels to nourish the tissue and cells2. The matrix then gradually organizes and the structure and integrity of

the injured area are restored. By an increase in the number of stem cells in the injured area, it is possible to increase

matrix deposition and therefore, accelerate the quality and rate of wound healing. This is the basic hypothesis that

has motivated the researchers to use stem cells for wound healing.

Stem cells can be divided based on their self-renewal and Potency38,39. Self-renewal is the ability to go through

numerous cycles of cell division while maintaining the undifferentiated state. While potency is the capacity

to differentiate into specialized cell types40 (Fig. 1 and 2). Based on the potency, stem cells can be divided into five

groups28,41. The first type is the totipotent stem cells37. These cells can differentiate into embryonic and extra-

embryonic cell types40 (Fig. 2). Such cells can construct a complete, viable organism19,42. These cells are produced

by fusion of an egg and sperm cell. The cells produced by the first few divisions of the fertilized egg are also

totipotent. Actually the potency of these cells is the highest among other stem cell types40. The second type is the

pluripotent stem cells43. These cells are the progenies of totipotent cells and can differentiate into almost all cells

(Fig. 1 and 2). The cells derived from any of the three germ layers are examples of this type19. The third type is the

multipotent stem cells37 which can differentiate into a number of cells, but only those of a closely related family of

cells28. The potency of these cells is much lower than the totipotent stem cells and lower than pluripotent stem cells.

The fourth type is the oligopotent stem cells. These cells can differentiate into only a few cells, such as lymphoid or

myeloid stem cells41. Finally, the fifth group is the unipotent cells13,29. The potency of these cells is extremely low so

that they can produce only one cell type, their own. They have the property of self-renewal, which distinguishes

them from non-stem cells29,44. Therefore, all types of stem cells have the ability of self-renewal but their potency is

different and depends on the source that they have arisen from28. There are many concerns in the stem cell therapy.

One concern is the risk that transplanted stem cells could transform to carcinogen cell lines and become cancerous if

cell division continues uncontrollably26,29,45,46.

6

Types of stem cells based on their source

1. Embryonic

These cells can be obtained from the epiblast tissue of the inner cell mass of a blastocyst or earlier morula stage of

embryos (Fig. 2). A blastocyst is an early stage embryo, approximately four to five days old in humans and has

about 30-160 cells. The embryonic stem cells are pluripotent and give rise to all derivatives of the three

primary germ layers i.e. ectoderm, endoderm and mesoderm. If a proper stimulation is induced, under an optimum

condition, they can be differentiated into more than 200 different cell types of the adult body37,42.

2. Fetal

The primitive stem cells located in the organs of fetuses are referred as fetal stem cells. Fetal stem cell is one of the

best sources for stem cells in veterinary medicine because all fetal tissues are composed of stem cells and without

any difficulty these cells can be harvested from the fetus. However, fetal stem cell in human medicine is faced with

ethical concerns and for this reason the researches in this area are limited42.

3. Adult

Adult or somatic stem cells can be found in children, as well as adults. Pluripotent adult stem cells are rare but can

be found in a number of tissues including umbilical cord blood18. For autogenous adult stem cells, there are three

accessible sources: 1) Bone marrow, which requires extraction by harvesting, that is, drilling into bone, typically

the femur or iliac crest, 2) Adipose tissue (lipid cells), which requires extraction by liposuction, and 3) Blood, which

requires extraction through pheresis, where blood is drawn from the donor, similar to a blood donation, passes

through a machine that extracts the stem cells and returns other portions of the blood to the donor15,27,29,47,48 (Fig. 1).

Bone marrow has been found to be one of the most reliable sources of adult stem cells which have been used in

treating several conditions31,39,41,47,49,50. The quantity of stem cells of bone marrow has been found to be declining

with age22,51. Most adult stem cells are multipotent and are generally referred to by their tissue origin (mesenchymal

stem cell (MSCs), adipose-derived stem cells (ADSCs), endothelial stem cell, dental pulp stem cell, etc.)22,37. Usage

of adult stem cells in research and therapy is not as controversial as the application of embryonic stem cells, because

production of adult stem cells does not require destruction of an embryo27. In addition, there is no risk of rejection in

instances where the adult stem cells are obtained as autografts38,46.

7

The pluripotent mesenchymal progenitor cells are denoted as stromal or MSCs26. Bone marrow contains two

main cell types: hematopoietic cells and stromal cells. The stem cells for non-hematopoietic tissues are referred as

MSCs because of their ability to differentiate as mesenchymal or stromal cells17. The mesenchymal cells are easily

obtainable from bone marrow by means of minimally invasive approach and can be expanded in culture and

permitted to differentiate into the desired lineage (Fig. 1). Differentiation can be achieved by application of

bioactive signaling molecules such as growth factors3,33. The transforming growth factor beta (TGF-beta) and bone

morphogenetic proteins (BMPs) are the most important factors of chondrogenic and osteogenic differentiation of

mesenchymal stem cells52. BMP 2, 4 and 7 may play important roles in chondrogenic and osteogenic

differentiation20,25,52. Czernik et al53 showed that sheep bone marrow-derived (BMD) MSCs, under appropriate

culture conditions, could be induced to differentiate into adipocytes, chondrocytes, and osteoblast lineages. Stem

cells can be seeded on the scaffolds for better drug delivery20. Many growth factors can also be assembled to the

scaffold to affect cell behavior54. Wodewotzky et al55 have successfully seeded canine BMD-MSCs on collagen

scaffolds and addition of basic fibroblast growth factor (bFGF) to the cell culture medium significantly increased the

proliferative rate of MSCs by 63% when compared to controls.

4. Amniotic

Multipotent stem cells are also found in amniotic fluid. These stem cells are very active, expand extensively without

feeders and are not tumorigenic. Amniotic stem cells can differentiate into adipogenic, osteogenic, myogenic,

endothelial, hepatic and also neuronal cell lines. Application of stem cells from amniotic fluid overcomes the ethical

objections of using human embryos as sources of cells. It is possible to collect amniotic stem cells for donors or for

autologous use18.

5. Cord blood

Recently, new source has been introduced for the autogenous stem cells. In fact, stem cells can be obtained from

umbilical cord blood just after birth. A certain kind of cord blood stem cell (CB-SC) is multipotent and displays

embryonic and hematopoietic characteristics. CB-SCs display very low immunogenicity because of expressing very

8

low level of major histocompatibility complex (MHC) antigens and failure to stimulate proliferation

of allogeneic lymphocytes. They can give rise to three embryonic layer-derived cells in the presence of

different inducers18.

6. Induced pluripotent

These are not adult stem cells, but rather adult cells (e.g. epithelial cells) reprogrammed to give rise to pluripotent

capabilities. Using genetic reprogramming with protein transcription factors, the pluripotent stem cells equivalent

to embryonic stem cells have been derived from human adult skin tissue38,56.

Role of stem cells in tendon healing (Tables 1 and 4)

Tendon healing is a slow process. Tendon has low vascularity and is under tension during the healing process. After

the injury, the cellularity is also not raised sufficiently to restore the structure and function of the tendon2,5-8.

Therefore, there is a need to increase the rate and quality of tendon healing. Stem cell therapy is one of the available

options with encouraging results2. Different stem cells have been used by many to promote tendon healing. Adult

stem cells are the most popular type; however aging reduces the potency of the stem cells57. Usage of growth factors

may be an important step in the tissue engineering of human flexor tendons. Raghavan et al58 showed that bFGF,

insulin-like growth factor (ILGF), and platelet-derived growth factor (PDGF) improve culture conditions for human

fibroblasts, tenocytes, and ADSCs and increase cellular proliferation and repopulation of a tendon scaffold in vitro.

One of the most scientifically tested stem cells is tendon-derived stem cells (TDSCs) that have recently been

identified within tendon tissues37. TDSCs exhibit universal stem cell characteristics, such as clonogenicity, a high

proliferative capacity, multi-differentiation potential, non-immunogenicity, and immunosuppression37. As a result,

implanting TDSCs in the injured area may be an effective way for tendon regeneration38,59. Ni et al60 used TDSCs to

treat the rat patellar tendon window (PTW) defect model. The TDSCs significantly improved collagen production,

cell and collagen alignment, and increased ultimate stress and Young's modulus compared to controls. Ni et al59

seeded the scaffold with TDSCs to produce neo-tendon. They implanted the cell seeded scaffold in a rat PTW injury

9

model and showed that it could produce tendon like tissue in vivo. In another study, Zhang and Wang61 showed that

implantation of engineered tendon matrix seeded with TDSCs, promoted tendon like tissue formation whereas

implantation of TDSCs alone did not lead to such tissue formation in vivo. These studies showed that scaffolds have

an important role in increasing efficacy of cells when cell therapy is a treatment strategy. The role of scaffolds as a

vehicle for stem cells should be highlighted. It is possible to change the behavior of these cells by changing the

architecture of the scaffolds. Yin et al62 showed that an aligned electrospun nanofiber scaffold provides an

instructive microenvironment for human TDSCs differentiation and may lead to development of desirable

engineered tendons both in vitro and in vivo. Although alignment of the scaffold is important but other factors such

as its dimension is also important for the stem cell behavior. Klatte-Schulz et al57 showed that 3D cell cultures are

more effective methods for stem cell culture and raising the cell activity compared to the 2D systems. Different

materials have been used as a scaffold for cell seeding. Collagen and silk are examples. Collagen has been shown to

have excellent biocompatibility and biodegradability. Shen et al44 used allogenous TDSCs-seeded knitted silk-

collagen sponge scaffold to treat rotator cuff tendon injury (RCTI) in rabbits. Application of such treatment strategy

did not elicit an immunological reaction, but instead increased fibroblastic cell ingrowth and reduced infiltration of

lymphocytes within the implantation sites at 4 and 8 weeks post-surgery. After 12 weeks, the treated group exhibited

an increased collagen deposition and had better structural and biomechanical properties compared to the control

group.

Different materials can alter the behavior of the tendon stem cells. Zhang et al63 showed that dexamethasone

suppress collagen type I expression and induce non tenocyte differentiation in vitro. Implantation of dexamethasone

treated human TDSCs in vivo, resulted in the extensive formation of fatty tissues, cartilage-like tissues, and bony

tissues. Such changes are not beneficial for tendon healing and therefore usage of such reagent should be inhibited.

In another way, Chen et al64 showed that autologous platelet-rich clot releasate can induce tenocyte differentiation,

proliferation, collagen types I and III and TGF-β1 concentration while suppressing the adipocyte, chondrocyte and

osteocyte lineages believed to impede tendon healing, in vitro. Klatte-Schulz et al57 also showed that BMP-2 and

BMP-7 are effective in increasing cell activity and collagen type I expression and protein synthesis with the more

effectiveness of BMP-7, in vitro.

10

Autologous skin-derived tendon-like cell (ASD-TLC) is another source for cell based therapy. The fibroblasts of the

skin have the same characteristics as the tenoblasts and tenocytes. Both of these tissues are soft connective tissue

and both of these cells produce collagen type I. Clarke et al65 in a randomized controlled trial study used ultrasound-

guided injection of ASD-TLC to treat the patients with lateral elbow tendinopathy. It improved the clinical

functionality of the patients and ultrasonographical characteristics of the tendons after 6 month follow up.

BMD-MSCs are another source of stem cells that has been used widely in treating tendon injuries. One possibility

is that the MSCs could be differentiated in vitro before in vivo application. As suggested, growth factors can be used

to achieve such goal. Schneider et al66 cultured MSCs in vitro and used growth factors IGF-1, TGF-β1, IGF-1/TGF-

β1, PDGF-BB, and BMP-12 to differentiate these cells. They showed that tenogenesis can be induced in MSCs

through a combination of treatment with IGF-1 and TGF-β1, in high-density co-cultures and through cultivation

with the spent media from primary tenocytes. However this is an expensive way. Although the other approaches

may theoretically be less effective than the first way; however some studies attempt different inexpensive ways. Yao

et al41 coated the sutures with rat BMD-MSCs to treat a rat Achilles tendon transection model. Stem cells were

present at all-time points (4, 7, 10, 14, and 28 days). The treated tendons showed greater strength at days 7 and 10

but not at other time points, compared to the control group. Also, Chong et al67 used BMD-MSCs to treat an

Achilles tendon transection model, in vivo and indicated that the treatment was not effective at least at gross

pathology. Cell labeling showed that MSCs were viable even after 6 weeks and diffused in the intra tendinous area.

Although treatment improved collagen fiber alignment and biomechanical properties of the injured area at 3 weeks

however at 6 weeks no significant difference were observed. It seems that MSCs has only a minor role and its role is

limited to the early stages of tendon healing.

Caniglia et al68 injected BMD-MSCs into the core defect in the superficial digital flexor tendon (SDFT) of horses.

They showed that intra lesional injection (ILI) of MSCs was not an effective manner to increase size of the collagen

fibrils as well as collagen density in horses. On the other hand, in a clinical study Godwin et al69 demonstrated that

ILI of MSCs is a safe technique in the tendinopathic SDFT and reduces rate of re-injury in the race horses. Although

the results of the Godwin et al69 may be encouraging but it should be highlighted that their method of assessment

was mainly based on the clinical history and they have not investigated neither morphology nor biomechanical

properties of the healing tendons and it seems, the conclusion of Caniglia et al68 who ultrastructurally investigated

11

the role of MSCs on tendon healing, is more reliable and scientific. It seems BMD-MSCs have low healing

promotive potential in tendon and this low effectiveness is not correlated with the route of administration (direct

injection vs. implantation of the cell seeded scaffold).

In another study, Pacini et al70 used undifferentiated MSCs to treat clinical SDFT injury in racehorses and indicated

that the injured area was healed and the fibers were ultrasonographically aligned and 9 of 11 horses returned to their

sport activity. They suggest that such a treatment strategy may be sufficient to repair damaged tendons without use

of scaffold support. They70 also showed that there is no need to differentiate the MSCs for treating tendon injuries at

least in horses. It is possible that aging of the differentiated MSCs have an impact on their effectiveness. It seems the

undifferentiated MSCs are younger than those of differentiated ones. The other reason for the effectiveness of

undifferentiated MSCs may be because the injured environment is enriched of growth factors and cytokines and

therefore, this environment may have some roles in differentiating these cells in vivo. However these statements

should further be investigated.

In another approach and in a simpler and inexpensive way, Okamoto et al43 treated Achilles tendon ruptures in a rat

model with transplantation of whole bone marrow cells. They hypothesized that bone marrow cells might

differentiate into regenerated tenocytes and presence of several cytokines within bone marrow cells might accelerate

tendon healing. Allologous bone marrow stem cells (BMSCs) were able to improve the ultimate failure load,

collagen production, and expression of TGF-β and VEGF in the treated lesions compared to those treated with

MSCs. They reported that the ultimate failure load of the treated lesions with BMSCs was comparable to the normal

tendons after only 28 days of injury. It seems other factors may contribute to these beneficial results. Except MSCs

other cells are also present in the bone marrow. Crovace et al71 compared the regeneration abilities of cultured

BMD-MSCs and bone marrow mononuclear cells (BMMNC) in collagenase-induced tendinitis of the

Achilles tendon in sheep. They showed, both cell types were able to improve collagen type I and matrix production

and increased the collagen alignment compared to controls. In another study, Crovace et al39 compared treatment

with cultured BMD-MSCs, BMMNCs, to repair collagenase-induced tendinitis in horses. Cell based treated lesions

showed higher expression of collagen type I, and cartilage oligomeric matrix protein and the fibers were more align

than the control lesions. Pascual-Garrido et al72 in a case series showed that after injection of autogenous BMMNC

in the injured area of patients engaged with chronic patellar tendinopathy the clinical outcome was excellent after 5

12

years follow up. All of these studies, however showed that BMMNC have more role in tendon healing and this may

explain why Okamoto et al43 that used whole bone marrow cells, have found better results than those tested MSCs

alone67,68.

Adipose derived stem cells are another type of mesenchymal stem cells. Several studies have suggested the

effectiveness of adipose derived MSCs (AD-MSCs) in tendon healing, in vivo. Uysal et al73 showed that ADSCs

could increase the tensile strength, collagen type I, FGF and VEGF levels of the treated tendons in vivo. Behfar et

al74 treated the experimentally induced transected deep digital flexor tendon of rabbits using intra tendinous injection

of ADSCs and showed that this treatment regimen caused significant increase in ultimate and yield loads, stress, and

energy absorption compared to the controls at 3 and 8 weeks after injury.

The major goal of regenerative medicine is to determine experimental techniques that take maximal advantage of

reparative processes that occur naturally in the injured area of animals. Injection of mesenchymal stem cells into the

core of damaged tendon represents such an approach. Acellularization of native tendons as potential targets and

seeding protocols are currently under investigation. Martinello et al75 recellularized a scaffold from cadaveric tissue

using AD-MSCs for use in total or partial tendon injuries. James et al76, also successfully seeded synthetic scaffolds

with the rat ADSCs in vitro and showed that these cells can be differentiated intro tenocyte like cells and produce

several tendon marker cells in vitro.

Blood derived MSCs (BD-MSCs) are another types of MSCs which are effective in healing. Daher et al77 surgically

repaired the simple Achilles tendon transection model of rats and covered the injured area, using biodegradable

scaffold seeded with allogenic BD-MSCs. They showed that using BD-MSCs as an adjunct in tendon repair

demonstrates superior biomechanical properties and an improved level of histological organization, when compared

to the control group. In another study, Marfe et al78 used BD-MSCs to SDFT injuries of the clinically ill race horses.

The race horses received a single autologous BD-MSC, which resulted in successful repair of the tendon. Both of

these studies showed similar results except that Daher et al77 used allogenous form but Marfe et al78 used autogenous

form of blood derived stem cells. It seems both the auto and allogeneic cells are effective in tendon healing in vivo.

Gene therapy is a new method of stem cell therapy. By this technology a specified gene is transmitted into genome

of the cells via a plasmid. This gene increases production of a specific protein, such as collagen or a growth factor,

13

which is necessary in tendon healing. Pelled et al52 implanted BMP2-engineered MSCs in a full-thickness defect of

the murine Achilles tendon. At 21 days, the treated tendons showed significantly enhanced biomechanical

performance and structural organization. However they failed to show whether the treatment strategy maintained the

superior biomechanical properties in a longer period of tendon healing (e.g. 120 days). Their results were not more

encouraging than those suggested the early effectiveness of MSCs alone, in vivo41,67.

Other types of stem cells are fetal and embryonic stem cells. Theoretically, implantation of such cells in the injured

area should initiate the tissue reaction and this host immune mediated inflammation should destroy the implanted

cells and retard their function. However, investigations represent a different conclusion. Watts et al79 used ILI of

male, fetal derived embryonic stem cells (ESCs) to treat SDFT defect model in horses. Application of ESCs

improved tissue architecture, tendon volume, and tendon linear fiber pattern. This technique was found to be safe

and well tolerated by the horses and did not increase inflammation at injured site. In another study, Cohen et al80

described the efficient derivation of connective tissue progenitors (CTPs) from the human embryonic stem

cells (hESC) lines and fetal tissues. The CTPs were significantly expanded and induced to generate tendon tissues in

vitro, with ultrastructural characteristics and biomechanical properties typical of mature tendons. They showed that

by this treatment strategy it is possible to successfully repair injured Achilles tendons and restore the ankle joint

extension movement in mice. They showed that CTP's have this ability to differentiate into bone, cartilage, and fat

both in vitro and in vivo.

Different types of stem cells such as MSCs, ESCs and TDSCs have been tested to improve tendon healing in vivo.

However the major limitation of previous studies was that they have not compared different cell types to show what

kind of stem cells are more efficient in tendon healing. Guest et al42 injected MSCs and ESCs into distinct areas of

damaged SDFT in horses and monitored their survival over a 3 month period. Neither MSCs nor ESCs produced

signs of cell-mediated immune response or tumor formation. ESC survival rate was high and cell count maintained

at a constant level over 90 days. ESCs were present at all sites of damage. In contrast, MSCs showed <5% survival

at 10 days and number of cells declined over the course of the experiment. MSCs were detected only at the site into

which they were injected. These results suggest that ESCs may be more effective in tendon healing than MSCs. In

another study, Pietschmann et al81 compared the potential healing ability of two different cell types in vivo. They

produced a critical full size Achilles tendon defect in rats and bridged the defect with polyglycolic acid and collagen

14

type I scaffold. The scaffold was seeded with MSC or TDSCs, while the control group was left without cells. After

16 weeks, samples loaded with tenocytes, had significantly higher biomechanical properties than the MSCs or

controls.

Role of Stem cells in Tendon to bone healing (Tables 2 and 4)

Fixation and incorporation of the ruptured rotator cuff tendon to bone is a major concern in rotator cuff repair

surgery. Rotator cuff repair usually fails at the tendon-bone interface, especially in case of large or massive tears.

Similar concern exists for the reconstructed ACL22,23. The torn ACL should be replaced by a new graft and the major

concern is the difficulty to fix the tendon graft into the bone tunnel22,23. The failure in most instances occurs at this

site22,23. In both cases, good fibrocartilageous zone should be developed and be mineralized to produce a strong

tendon to bone attachment. Stem cells are used to improve the quality and rate of tendon to bone healing22-24,40,82,83.

Nourissat et al40 explored the benefits of a treatment based on injecting chondrocyte and MSCs in a rat model of

degenerative enthesis repair. The Achilles' tendon was cut and the enthesis destroyed. The damage was repaired by

classic surgery. Cell injection significantly improved healing and the load-to-failure after 45 days and it also

produced new enthesis in the treated lesions.

Although, application of stem cells alone may have some effective roles in tendon to bone healing however, some

studies have suggested that there is a need in inducing osteogenesis at this site so that the stem cells should be

activated to produce bony structure. Studies revealed that stem cell differentiation may have more value in tendon

to bone healing than tendon healing. Silva et al84 showed that adult non-cultivated BMD-MSCs did not

accelerate tendon-to-bone healing in the femoral tunnel, after hamstring ACL reconstruction in humans. Similarly,

Gulotta et al49 used allologous BMD-MSCs to treat rotator cuff tendon injury in rats. Addition of MSCs to the

healing rotator cuff insertion site did not improve the structure, composition, or strength of the healing

tendon attachment site. These disappointing results may be because the repair site may devoid of the cellular and/or

molecular signals which are necessary to induce appropriate differentiation of transplanted cells. Rui et al85 showed

that BMP-2 can differentiate the TDSCs so that it promotes the osteogenic, adipogenic, and chondrogenic

differentiation but inhibit tenogenic differentiation in vitro. To assemble such growth factor with stem cells, a

15

scaffold is needed. In addition, it seems the scaffolds have some beneficial role in tendon-bone healing. Yokoya et

al86, seeded a polyglycolic acid sheet scaffold with MSCs and reconstructed infraspinatus tendon defect model in

rabbits and indicated that MSCs were able to regenerate tendon-bone insertions and the tendon belly, and increased

production of type I collagen, and mechanical strength of the regenerated rotator cuff tendon. Soon et al23 also

analyzed the effect of coating allografts with MSCs on ACL graft healing after ACL reconstruction in rabbits. The

treated group showed a mature zone of fibrocartilage blending from bone to the allograft, strongly resembling a

normal ACL insertion. MSCs treated lesions also showed higher ultimate load but lower stiffness and Young's

modulus compared to the control lesions. In a similar study Lim et al22 studied the effect of coating tendon grafts

with MSCs on the rate and quality of graft osteointegration in ACL reconstruction in a rabbit model. Treatment

increased the quality of fibrocartilage zone histologically and higher amount of collagen type II was seen in these

lesions. Also after 8 weeks, the treated lesions showed higher ultimate load and stiffness than controls.

Based on the above evidences it seems the role of scaffolds in tissue healing, which results in enhanced effectiveness

of seeded stem cells should be highlighted. As suggested, an optimum tissue engineered product is composed of

scaffold, growth promotive factor and stem cell. Such a construct is used to repair the tendon bone healing in vivo.

Chen et al83 enhanced infraspinatus tendon-bone healing, using an injectable hydrogel made with rabbit periosteal

progenitor cells and polydiacrylate tethered with BMP-2. Treatment improved production of collagen type II,

aggrecan and fibrocartilage tissue. It also increased ultimate strength of the tendon-bone interface after 4 and 8

weeks.

Some others tried gene therapy to induce healing. Dong et al87 used bone marrow stromal cells and infected these

cells with recombinant BMP-2 virus to reconstruct ACL in rabbits and showed that treated samples had higher

ultimate load and stiffness and better collagen fiber alignment than controls. Scleraxis is a basic helix-loop-helix

transcription factor that is thought to lead tendon development during embryogenesis. Gulotta et al56 determined that

application of MSCs transduced with adenoviral-mediated scleraxis could improve regeneration of the tendon-bone

insertion site in a rat rotator cuff repair model. Scleraxis improved fibrocartilage production with improved collagen

fiber organization and increased tensile strength.

Use of different types of stem cells with lower potency, but in a more differentiation status has also been reported.

Mifune et al24 showed that the ACL-derived CD34(+) stem cells could contribute to tendon-bone healing in rats.

These cells increased collagen production, angiogenesis and osteogenesis in vivo. In another study, Ju et al82

16

investigated the role of synovial MSCs on tendon-bone (Achilles tendon was fixed within the tibial bone tunnel

experimentally) healing in rats. Cell therapy increased proportion of collagen fiber area and sharpey’s fibers and the

cells were migrated in the tendon to bone junction during the first two weeks but the labeled stem cells were no

longer observable after four weeks.

The effectiveness of stem cells in tendon to bone healing has been also reported in a limited number of clinical

studies. Ellera et al88 in a case series treated rotator cuff tears with conventional surgical technique but with aiding

autologous BMD-mononuclear cells. They reported that the integrity of tendon was improved by the cells and the

patients had proper outcome after one year follow up.

Role of stem cells in bone regeneration and healing (Tables 2and 4)

Bone healing has its own limitations and complications. Many cases of nonunion, malunion or osteomyelitis are

presented yearly89-91. In addition in such cases associated with large massive bone defects, such as osteosarcoma,

gunshot fractures, severe trauma, burn, etc, bone transplantation is needed, however a proper graft both in size and

quality may not be available for such cases. Therefore, there is a need to accelerate bone healing to increase the

amount of the newly regenerated callus in the defect area. Stem cells may have a role to aid bone formation in this

regard. Several studies have been conducted in vitro and in vivo to test whether stem cell therapy is effective in

achieving such a goal12,14-16,18-21.

The osteogenic potential of MSCs has been previously suggested92. Also, it has been shown that this potential is

stronger in autogenous MSCs. Niemeyer et al16 used autologous ovine BMD-MSCs and xenogenic based human

BMD-MSCs, cultured on mineralized collagen scaffold and implanted into a 3.0-cm-long sheep tibia bone defect.

Compared to the human cells, the autologous cells resulted in improved bone healing. However, the human cells

were present in the defect area even after 26 weeks of cell transplantation in vivo. There are also some evidences that

confirmed the above results and indicated the effectiveness of MSCs in bone healing. Ai et al19 compared

the bone healing rate after a human xenograft of mineralized bone and together with an allograft of BMD-MSCs in

tibial bone defect of a rabbit model. Stem cells produced more bone like tissue in the defect area with less

inflammation than controls. In some studies MSCs have been injected into the injured area. Shao et al13 injected the

17

BMD-MSCs to the experimentally induced gap of distracted callus in rabbits. The stem cells were able to increase

bone union of the distracted regenerate site compared to the control. Treatment increased bone formation and bone

density in distraction site. Obermeyer et al17 used allologous BMD-MSCs to treat alcohol induced healing

impairment in a mid-shaft tibia fracture model in mice. Treatment restored both fracture callus volume and

biomechanical strength of the injured area. In vivo imaging demonstrated a time-dependent MSC migration to

the fracture site. Giannotti et al91 in a case series used BMD-MSCs to treat the upper limb fractures that shown

pseudo-arthrosis and delayed of consolidation. Four women and four men were followed with a follow-up of 50.3

months. In all cases the site of non-union had been revitalized, by microfractures and drilling, and a synthesis was

performed with a rigid plate. So they filled the bone gap with autologous bone and MSCs expanded in the

laboratory. All the eight cases healed and this was confirmed radiographically.

There are some evidences that BMD-mononuclear stem cells are also effective in bone healing. Rickert et al93 in a

controlled trial study, reconstructed the atrophic maxilla with a bilateral sinus floor augmentation procedure. On one

side the augmentation procedure was performed with bovine bone mineral seeded with mononuclear stem

cells harvested from the posterior iliac crest (test group) while bovine bone mineral mixed with

autogenous bone was applied on the contralateral side (control group). After 3 month the mononuclear stem cells

produced more bone formation at histology.

Other sources of MSCs have also been used to accelerate bone healing. Lendeckel et al94 used autologous

ADSCs to treat the clavarial defect in a 9 year old girl. Mechanical fixation was achieved by two large, resorbable

macroporous sheets acting as a soft tissue barrier at the same time. The postoperative course was uneventful and CT-

scans showed new bone formation and near complete calvarial continuity three months after the reconstruction. In

another study, Hao et al15 fabricated a biomimetic construct based on a combination of rabbit ADSCs encapsulated

in collagen I gel with a PLGA-beta-TCP scaffold. The composites were implanted into a 15-mm length critical-sized

segmental radial defect. After 24 weeks, the medullary cavity recanalized, bone was rebuilt, and molding finished,

the bone contour remodeled smoothly and the scaffold degraded completely in the treated group.

One of the other sources of MSCs is periosteum. Funk et al89 used periosteum-derived MSCs in a patient with

atrophic non-union of the distal femur after correction osteotomy. Their radiographical results indicated that

treatment promoted bone healing in the defect area. Other sources of MSCs, also have been introduced. Qu et al90

investigated the clinical effect of human cord blood MSCs on bone nonunion of the femoral and tibial fracture.

Treatment accelerated bone healing. In another study, Uchida et al18 used bone marrow-engrafted umbilical cord

blood-derived MSCs to treat fracture injury in mice. The cells differentiated into both the hematopoietic and

mesenchymal cells and induced healing of the defect area.

The most standard methods of stem cell therapy are direct injection or cell seeding (cell + scaffold) and

transplantation of a graft. However different ways were tried by some. For example, Lee et al14 investigated an in

vivo life cycle of ADSCs in an animal model of skeletal injury. Primary stem cells were lentivirally transfected with

a fusion reporter gene and injected intravenously into mice with bone injury or sham operation. They showed that

systemically administered stem cells migrated to the site of skeletal injury and facilitated bone healing.

Different methods have been conducted to differentiate the stem cells in order to increase their osteogeniec potential

Ozturk et al12 used bone marrow MSCs as the source of osteo-progenitor cells and stimulated them with

prostaglandin E2 (PGE2), using demineralised bone matrix as a carrier. They treated rats with segmentary

radial bone defects by such a treatment modality. The PGE2 stimulated stem cell differentiation into the osteoblastic

cells and improved bone healing. As stated, BMP4 is one of the main local contributing factors in callus formation.

Lin et al95 showed that when ADSCs was exposed to adenovirus containing BMP4 cDNA could differentiate into

osteoblasts and produce bony tissue, both in vitro and in vivo. PRP contains a mixture of growth factors that play an

important role in soft and hard connective tissue healing3. Kasten et al96 showed that PRP may have a role on MSCs

differentiation into osteoblasts however, they stated that the differentiation status of the MSCs and the type of the

scaffold which MSCs are seeded on, are crucial factors. Some clinical evidences support this hypothesis that PRP or

its growth factors may be beneficial in stem cell therapy. Behnia et al97 evaluated the enhancing effect of

recombinant PDGF on human MSCs in secondary alveoloplasty. They seeded a biphasic scaffold with human MSCs

and reached the construct with PDGF as a growth factor to provide all three necessary constitutions of a standard

tissue engineered graft. The graft were placed in anterior maxillary cleft defects of three patients and closed with

lateral advancement gingival flaps. The postoperative cleft bone volume was measured with cone beam CT scans. A

mean of 51.3% fill of the bone defect was calculated 3 months post-operation.

Recently, simvastatin has been shown to have some role in MSCs behavior in vivo. Qi et al20 investigated the effects

of simvastatin locally applied from calcium sulfate combined with a MSC sheet on fracture healing of an osteotomy

model in a rat tibia. At two weeks, treatment significantly increased expressions of BMP2, alkaline phosphatase,

19

osteocalcin, osteoprotegerin and VEGF, with more callus formation around the fracture site compared to controls.

After 8 weeks, complete bone union was obtained in the cell treated group however the control groups showed no

bone union at this time.

It is suggested to use combined treatment modalities to increase the effectiveness of MSCs in bone healing. Many

methods exist and an example is provided here. Cheung et al21 investigated the effects of combined treatment of

exogenous MSCs and low intensity pulsed ultrasound on fracture healing in rats. Treatment accelerated bone

healing; however the MSCs had the major role in this regard.

Role of stem cells in cartilage healing and osteoarthritis (Tables 3 and 4)

The incidence of cartilage injury and osteoarthritis are high30,32,46,50. Cartilage as tendon has minimum healing

capability and in degenerative conditions such as OA may not be healed and progression of abrasion injury usually

develops to completely destruct the tissue. Injured cartilage should tolerate strong weight bearing forces and wear

and tear stresses facilitate this destruction. Pain is the most significant signs of OA but some other signs such as joint

stiffness, edema and loss of range of motion could be developed by time. The outcome of OA or cartilage injury, if

neglected, is poor. Therefore, it is important to find a way to induce and increase cartilage regeneration and healing

and also to inhibit OA progression in all cases. Stem cells have been shown to have beneficial roles in this

regard28,29,51,99,98.

It has been suggested that MSCs therapy can increase the rate and quality of cartilage regeneration both in animals

and humans. Zscharnack et al28 used autologous pre-differentiated MSCs embedded in a collagen I hydrogel to treat

chronic osteochondral defect in ovine stifle (knee) joint. Treatment improved histologic scores with morphologic

characteristics of hyaline cartilage such as columnarization and presence of collagen type II. In another study, Horie

et al45 investigated whether intra-articular (IA) injection of synovium-MSCs enhances meniscal regeneration in rat

massive meniscal defects. After 12 weeks the cells adhered to the lesion, differentiated into meniscal cells directly,

and promoted meniscal regeneration without mobilization to distant organs. The cells also increased collagen type II

production. Kasemkijwattana et al50 reported the results of autologous BMD-MSCs implantation in two patients

with large traumatic cartilage defects of the knee. After 30 month, no post-operative complication was noted and the

20

patients' clinical scoring improved by time. The arthroscopic assessment showed good defect filling, stiffness and

incorporation to the adjacent cartilage. Connell et al100 in a prospective clinical pilot study to treat patients with

refractory lateral epicondylitis injected collagen-producing cells derived from dermal fibroblasts into the sites of

intrasubstance tears and fibrillar discontinuity of the common extensor origin under ultrasonography guidance. They

reported that the pain and function as well as the quality of the healing tendons were improved. Orozco et al101

treated the lumbar disc degeneration with intact annulus fibrosus using autologous expanded BMD-MSC injected

into the nucleus pulposus area. Treatment improved pain and disability and the outcome was favorable after 1 year

follow up. Wakitani et al46 also used human autologous MSCs to treat the joint injuries. They stated that among 41

patients, neither tumors nor infections were observed between 5 and 137 months of follow-up.

BMP2 and PRP have been shown to be effective in increasing the ability of stem cells to produce cartilage tissue.

Zachos et al25, tested whether stem cells transduced with BMP2 can promote cartilage repair. Distal femoral articular

osteotomies in nude rats were treated with MSCs, either wild-type or transduced with an adenoviral BMP2. After 14

days, only the group that received BMP2-stem cells by direct injection showed completely healed osteotomies and

these cells improved the healing quality of the bone and cartilage in vivo. PRP is reported to promote collagen

synthesis and cell proliferation as well as enhance cartilage repair3. Mifune et al54 showed that PRP could enhance

the therapeutic effect of muscle derived stem cell therapy for the OA treatment in rats. Addition of PRP to the stem

cells could increase BMP-4 expression which is important for cartilage regeneration. PRP also enhanced collagen

type II production, stem cell attachment, migration and proliferation in vivo.

As stated, OA is a complicated condition that should be controlled as soon as a time of diagnosis99 102. Centeno et

al103 in a review study has suggested that percutaneous injection of MSCs into a knee with symptomatic and

radiographic OA, resulted in significant cartilage growth, decreased pain and increased joint mobility. There are

some in vivo and clinical evidences represent that stem cell therapy may reduce signs of OA. Such treatment strategy

also has been shown to increase the chance of cartilage regeneration in this situation. Different types of MSCs have

been investigated in OA based studies. ter Huurne et al29 determined the effect of IA injection of ADSCs on

synovial lining thickness and its relation to joint pathology in a mouse model of OA. The stem cells inhibited

synovial thickening, formation of enthesophytes associated with ligaments, and cartilage destruction. In another

study, Sato et al98 tested the outcome of IA transplantation of hMSCs suspended in HA in the knee joints of OA

21

induced guinea pigs. After 5 weeks, the xenogenic based hMSCs improved the quality of the repaired cartilage and

the cells well migrated, proliferated and differentiated in the OA cartilage. Rabbit is a better model for studying OA

than mouse and guinea pigs. Grigolo et al27 transplanted in a rabbit model of OA a hyaluronan-based scaffold seeded

with BMD-MSCs. After 3 and 6 months, the stem cells reduced the morphological signs of OA. In a larger animal

model, Al Faqeh et al51 determined whether an IA injection of a single dose of autologous chondrogenic induced

BMD-MSCs could retard the progressive destruction of cartilage in a surgically induced OA in sheep. Six weeks

after injection, the cells improved cartilage quality and reduced OA progression. In a clinical study in dogs with hip

OA, Black et al104 showed that the dogs treated with ADSCs gained better scoring for lameness, pain, and range of

motion than controls. Similar study was performed by Black et al26 in doges with chronic OA of the humeroradial

(elbow) joints and similar results were recorded. In another study, Jiang et al99 evaluated the repair potential of the

selected chondrogenic clonal MSCs by direct delivering them into the injured cartilage site in a collagenase-induced

OA model in Cynomolgus monkeys. After 8-24 weeks, the abrasions of articular cartilage were significantly

improved and repaired by MSC-based treatment. The treated lesions got better histologic scoring than the controls.

OA signs also were improved.

Clinical findings also support the results of the animal studies. Emadedin et al31 investigated the potential of IA

injection of autologous MSCs in six OA patients. During one year follow up, such a treatment modality was found

to be safe with no significant adverse effect. All the patients were partly satisfied with the results of the study. Stem

cell therapy improved pain, function of the knee, and walking distance. MRI results showed an increase in cartilage

thickness, extension of the repair tissue over the subchondral bone and a considerable decrease in the size of

edematous subchondral patches in half of the patients (3/6). In another study, Davatchi et al30 examined whether

MSC transplantation could reverse the OA process in the knee joint. Four patients with knee OA were treated with

IA injection of autologous MSCs. Cell therapy improved pain, walking time and crepitus. Minor improvement for

the range of motion was evident in the patients. Finally, Koh et al32 evaluated the clinical and imaging results of

patients who received IA injections of autologous ADSCs for treatment of knee OA. Treatment reduced pain and

improved knee function.

22

Discussion

This study comprehensively introduced stem cells as one of the treatment option in soft and hard connective tissue

injuries. We showed that autologous MSCs are the most popular cells that widely tested in different species with

encouraging results17,20,22,23,41,56,69,86,98. Although it seems that other types of stem cells are also effective in tissue

healing and regeneration however the studies are insufficient to prove their efficacy16. Different sources of MSCs

have been used by many investigators; however the most reliable sources seem to be both bone marrow and adipose

tissues. Generally, cell therapy was found to be a safe method even with the allologous (e.g. embryonic stem cells)

or xenologous forms42. Based on the reviewed reports, no adverse effects related to cell therapy were diagnosed16.

Despite of improvement in the tissue engineering technologies and presence of a large variety of scaffolds,

commercially available in the market and or can easily be made in a laboratory, however cell seeding technology

seems to be technically demanding and most of the studies, selected the direct injection method especially in the

clinical studies rather than cell seeding30-32,50,94. However, it has been shown that tissue engineered based cell

therapy has a superior potential in healing of injured tissues. This review also, showed that it is possible to

differentiate stem cells with healing promotive factors such as BMP and this differentiation is well achieved in an in

vivo condition. It seems in vitro cultivation and differentiation of the cells increase cell aging and decrease its

healing efficacy25,68,70-72. Therefore, a proper design for such treatment modality should be based on three factors

including, scaffold, healing promotive factors or cell differentiating agents, and stem cells. For better explanation,

the stem cells should be seeded on a scaffold in laboratory without altering its differentiation2,19. At time of

implantation of the graft the differentiating factor should be added to the construct. This inhibits cell aging and

increase the healing efficacy of the cells in the injured area. However, this conclusion was based on the presented

results; perhaps, future studies could better clarify this phenomenon.

It is impressive and also interesting that clinical studies, support experimental animal studies30,32,50,65,89,90,94, however,

several limitations exist that makes it hard to conclude. The first limitation is that, there are some controversies

between the results of some studies. For example, theoretically autogenous MSCs should have higher viability in the

injured area than the embryonic cells. However, some reports exist that ignore this statement39,42,49,68,70. This

potentially could be an area of research for future works. There are also some studies that have not followed an

standard pattern, and each had its own hypothesis and used different types of stem cells with different preparation

23

methods and cell concentration26,51,67,82,99. Another limitation is the lack of human clinical trials studies.

Unfortunately, most of the recent clinical based studies are case reports or case series which shows that cell therapy

is still in its primitive stages and is still under investigation (Table 4). The proportion of animal studies is extremely

higher in this regard. Although the animal based studies mimic the human condition at least as compared with in

vitro investigations, however it should be remembered that the animal studies are an approximation and each animal

model has its own limitations and benefits. Potential limitations of the animal studies should be considered when the

results are going to be generalized in clinical setting.

Conclusion

Cell therapy although has been shown to be an effective treatment in different tissue injuries, however there is no

uniformity between the methods and no standard method has been selected as a gold standard. There are numerous

animal studies which have tested these cells in different tissue injuries, however controversies exist and human

clinical studies are mainly limited to case reports and case series and lack of well-designed clinical trial studies in

this area of research gives this conclusion that cell therapy although interesting but is still in its primitive period.

BMD-MSCs are the most approved stem cell type and direct injection of the stem cells are the most reliable

technique. Future researches should focus on the role of tissue engineering in this regard with a better fusion

between tissue engineering technologies and in vivo application.

Conflict of Interest

The authors have no conflict of interest or financial discloser to persons and organizations. The authors received no

found for this work.

24

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36

Table 1: Role of stem cell therapy on the treatment of tendon injuries in animal studies

Reference Subject Model of injury Treatment modality Methods of assessments Main results Uysal et al73 Rabbit Achilles transection

and anastomosis ADSCs Immunohistochemistry,

biomechanics Increased tensile strength, collagen type I, FGF and VEGF levels

Yao et al41 Rat Achilles transection and anastomosis

BMD-MSCs Histology, biomechanics Increased biomechanical strength at early healing, not effective in later stages

Pelled et al52 Murine Achilles transection and anastomosis

Smad8/BMP2-engineered MSCs (Gene therapy)

Histology, biomechanics Improved biomechanical properties and structural organization of the repaired tendons

Marfe et al78 Horse SDFT injury BDSCs Clinical tests Improved tissue regeneration and function of the horses Shen et al44 Rabbit Rotator cuff tendon

injury TDSCs Histology, biomechanics,

biochemistry Increased collagen deposition and improved structural and biomechanical properties

Behfar et al74 Rabbit Deep digital flexor tendon transection and anastomosis

ADSCs biomechanics Improved biomechanical properties of the injured tendons

Caniglia et al68 Horse SDFT core defect MSCs Transmission electron microscopy Was not effective in increasing collagen fibrils diameter and density Ni et al60 Rat rat

Patellar tendon window defect model

TDSCs Histology, florescent imaging, biomechanics

improved collagen production, cell alignment, collagen fiber alignment and increased ultimate stress and Young's modulus of the healing tissue

Godwin et al69 Horse Tendinopathic SDFT MSCs Ultrasonography scintigraphy, histology

No abnormal tissue was formed and the rate of reinjury was decreased

Watts et al79 Horse SDFT defect Intra-lesional injection of male, fetal derived embryonic-like stem cells

Ultrasound, MRI, biochemical assays, gene expression and histology

Improved the tissue architecture, tendon size, tendon lesion size, and tendon linear fiber pattern of the treated tendons

Okamoto et al43

Rat Achilles tendon injury Bone marrow cells Immunohistochemistry, histology, biomechanics, molecular tests

Improved biomechanical properties of the treated lesion and it were comparable to normal tendons Also compared to the MSCs, bone marrow cells increased collagen production and gene expressing for the growth factors (VEGF and TGFB-2)

Guest et al42 Horse Mechanically induced SDFT injury

ECSs vs Autologous MSCs

Histology Embryonic stem cells had higher survival rate and better migration pattern than the mesenchymal had No tumor formation or immune reaction was evident in the injured area

Crovace et al71 Sheep Collagenase-induced tendinitis

Bone marrow stromal cells and, bone marrow Mononucleated Cells

Histology and immunohistochemistry Improved collagen fiber alignment and increased the expression of matrix component and collagen type I but decreased expression of collagen type III

Chong et al67 Rabbit Achilles tendon transection model and surgical repair

MSCs Histology, Immunohistochemistry, biomechanics, gross morphology

Was not effective however the stem cells were alive after 6 weeks of injury

Crovace et al39 Horse Collagenase-induced tendinitis

Bone marrow stromal cells and, bone marrow Mononucleated Cells

Molecular tests and histology Improved collagen fiber alignment and increased the expression of matrix component and collagen type I but decreased expression of collagen type III

Daher et al77 Rat Achilles tendon transection model and surgical repair

Circulatory stem cells Histology, biomechanics Improved collagen alignment, collagen quantity, increased ultimate load and stress of the treated lesions compared to controls

Gulotta et al56 Rat Rotator cuff tendon injury

MSCs transduced with adenoviral-mediated scleraxis

Histology, biomechanics Improved collagen organization, fibrocartilage production at bone tendon interface and increased ultimate strength and stiffness of the healing tissue compared to controls

Gulotta et al49 Rat Rotator cuff tendon injury

BMD-MSCs Histology, biomechanics Treatment was not effective

Pacini et al70 Horse Clinical SDFT injury Undifferentiated MSCs Ultrasonography, clinical tests Function of the animals improved, the collagen fibers were aligned under ultrasonography, no ectopic bone or tumor formation was seen in the healed lesions

Yokoya et al86 Rabbit Infra spinatus tendon defect reconstruction

MSCs Histology, biomechanics Improved collagen production and biomechanical properties

37

Table 2: Role of stem cell therapy on the treatment of bone and tendon to bone injuries in animal studies

Reference Subject Model of injury Treatment modality Methods of assessments Main results Bone healing Ozturk et al12 Rat Segmentary

radial bone defects Bone marrow stem cells +PGE2

Radiology, histology PGE2 differentiated bone marrow cells into osteoblasts and improved bone healing

Shao et al13 Rabbit Distraction of the

osteotomy site of mandible

Osteoblast like cells Histology, radiography, SEM, dual-energy x-ray absorptiometry.

Increased the bone formation at distraction site compared to the control site.

Lee et al14 Mouse Bone defect model Systemic

administration of ADSCs

Bioluminescence imaging, micro-PET, micro-CT

systemically administered stem cells migrated to the site of skeletal injury and facilitate bone healing

Hao et al15 Rabbit Radial bone defect ADSCs Histology, radiographs, bone mineral density, mechanical testing.

The cells improved the quality and rate of bone healing and increased scaffold degradation

Niemeyer et al16

Sheep Tibia bone defect Human and ovine BMD-MSCs

Radiology, histology Autologous stem cells were more effective and xenologous ones however the human cells were present in the healing area even after 26 weeks of injury

Obermeyer et al17

Mouse Alcohol induced osteotomy wound impairment

MSCs Biomechanics, histology, and micro CT

Cell therapy increased bone volume and biochemical properties of the healing tissue

Uchida et al18

Mouse Bone defect Bone marrow-engrafted umbilical cord blood-derived MSCs

Immuno histochemistry, histology

The stem cells differentiated into both the hematopoietic and mesenchymal cells and have role to heal the defect area by producing bone like tissue.

Ai et al19 Rabbit Tibial bone defect Xenograft scaffold seeded with BMD-MSCs

Histology, biomechanics Treatment produced more bone like tissue qualitatively

Qi et al20 Rat Osteotomy model in

tibia MSCs RT-PCR, x-ray, micro-CT,

histology Increased expressions of BMP 2, alkaline phosphatase, osteocalcin, osteoprotegerin and VEGF, callus production and induced complete bone union after 8 weeks

Cheung et al21

Rat Osteotomy model MSCs and ultrasound Radiography, Micro-CT Histomorphometry

MSCs had the major role in bone healing. These cells increased bone remodeling and increased the bone volume in the defect area

Tendon to bone healing

Lim et al22 Rabbit ACL reconstruction MSCs Histology, biomechanics Improved quality of the fibrocartilage zone at tendon to bone junction and increased collagen type II, ultimate load and stiffness.

Soon et al23 Rabbit ACL reconstruction MSCs Histology, biomechanics Improved the quality of the fibrocartilage zone and increased ultimate load but decreased stiffness and Young's modulus compared to controls

Ju et al82 Rat Achilles fixed into the tibial bone tunnel

Synovial MSCs Histology Cell therapy increased the proportion of the collagen fibers and improved tendon to bone attachment

Nourissat et al40

Rat Degenerative enthesis

Chondrocytes stem cells and MSCs

Histology, immunostaining and biomechanics

Stem cells produced new enthesis and improved biomechanical properties and healing rate of the treated lesions compared to the control ones.

Chen et al83 Rabbit Infraspinatus tendon-bone

periosteal progenitor cells

Histology, immunohistochemistry, biomechanics

Improved production of collagen type II, aggrecan and produced fibrocartilage tissue. It also increased ultimate strength of the treated tendon-bone interface

Mifune et al24

Rat ACL reconstruction ACL-derived CD34(+) stem cells

Immune histo chemistry, histology, and molecular tests

Increased collagen production, angiogenesis and osteogenesis

38

Table 3: Role of stem cell therapy on the treatment of cartilage injuries and osteoarthritis in animal studies

Reference Subject Model of injury Treatment modality

Methods of assessments Main results

Zachos et al25

Rat Distal femoral articular osteotomies

Stem cells transduced with BMP2

Gene expression and in vivo imaging

BMP2-cells were able to better affect the healing of bone and cartilage compared to the stem cells alone

Black et al26

Dog Chronic osteoarthritis of the humeroradial

ADSCs Clinical scoring cell therapy improved signs of lameness, pain on manipulation, range of motion, and functional disability in dogs

Grigolo et al27

Rabbit Osteoarthritis Hyaluronan-based scaffold seeded with BMD-MSCs

Histology, histomorphometry, immunohistology

Stem cell therapy reduced morphological signs of OA but was failed to inhibit OA progression

Horie et al45

Rat Massive meniscal defect

Synovium-MSCs Histology, transmission electron microscopy, luminescence analysis

Stem cells differentiated into meniscal cells directly, and promoted meniscal regeneration without mobilization to distant organs. The cells also increased collagen type II production.

Zscharnack et al28

Ovine Chronic osteochondral defect

Pre-differentiated MSCs embedded in a collagen I hydrogel

Histology Improved morphological characteristics of the regenerated cartilage in the defect area

Sato et al98 Guinea Pig

Osteoarthritis Human MSCs Fluorescent microscopy, histology, immunohistochemistry

Improved cartilage regeneration and the human stem cells well migrated, differentiated and proliferated in the cartilage matrix.

ter Huurne et al29

Mouse Osteoarthritis ADSCs Histology Stem cells inhibit synovial thickening, formation of enthesophytes associated with ligaments, and cartilage destruction.

Al Faqeh et al51

Sheep Osteoarthritis Autologous chondrogenic induced BMD-MSCs

Histology, Gross morphology

Reduced progression of OA but was not significant compared to the sham group. Cartilage and meniscal regeneration was better in the cell treated group.

Jiang et al99 Monkey Osteoarthritis Chondrogenic clonal MSCs

Clinical parameters, radiography, histology, immunohistochemistry

The abrasions of articular cartilage were significantly improved and repaired by MSC-based treatment. Function of the treated animals was better and the OA signs diminished significantly.

Mifune et al54

Rat Osteoarthritis PRP+ muscle derived stem cell

In vitro assays, molecular and biochemical tests, histology

PRP enhanced cell attachment, migration and proliferation, collagen type II production, BMP-4 expression by the stem cells

39

Table 4: Role of stem cell therapy in human clinical studies

Reference

Subject Number of cases

Model of injury Treatment modality

Follow up time

Methods of assessments

Main results

Lendeckel et al94

Case report 1 Clavarial defect Autologous ADSCs

3 month CT-scan Produced new bone formation and the defect was going to be closed.

Funk et al89

Case report 1 Atrophic non-union of the distal femur after correction osteotomy

Periosteum-derived MSCs

3 month Radiology Improved bone healing

Kasemkijwattana et al50

Case report 2 Traumatic cartilage defects of the knee

BMD-MSCs 30-31 month

Clinical scoring, arthroscopy

Cartilage defect were filled with new cartilage and the clinical scoring were improved by time

Behnia et al97

Case series 3 Alveolar bone defect MSCs + scaffold + PDGF

3 month CT scan About 50% of the defect area were filled with new bone

Davatchi et al30

Case series 4 Osteoarthritis BMD-MSCs 1 year Clinical tests Improved pain, walking time, crepitus, but not range of motion. The results were not excellent

Emadedin et al31

Case series 6 Osteoarthritis Bone marrow derived MSCs

1 year Clinical scoring, MRI

Stem cell therapy improved signs of pain, function of the knee and increased cartilage regeneration and reduced edema

Pascual-Garrido et al72

Case series 8 Chronic patellar tendinopathy Bone marrow mononuclear cells

5 years Clinical scores, ultrasound

Clinical signs were improved in 7 of 8 patients

Orozco et al101

Case series 10 Lumbar disc degeneration with intact annulus fibrosus

BMD-MSCs 1 year Clinical, MRI Signs of degeneration and pain were improved and the functionality of the patients restored

Connell et al100

Case series 12 Lateral epicondylitis Collagen-producing cells derived from dermal fibroblasts

6 month Clinical, ultrasonography

Treatment reduced pain and improved function, angiogenesis and quality of the healing tendons

Wakitani et al46

Case series 41 Joint injury BMD-MSCs 75 month History taking No infection of tumor formation was evident in any of the patients. The method was found to be safe.

Rickert et al93

Controlled trial

12 Atrophic maxilla Mononuclear stem cells

3 month Histology Stem cells increased the bone formation

Ellera et al88

Case series 14 Rotator cuff tears Autologous bone marrow mononuclear cells

1 year MRI, Clinical scoring

The integrity of the tendons improved and patient outcome was acceptable

Giannotti et al91

Case series 8 Psuodoarthrosis and non-healing bone fracture

MSCs expanded in laboratory

59 month Radiography All of the fractures were healed.

Koh et al32 Case series 18 Osteoarthritis ADSCs 24.3 month Clinical tests, MRI, radiography

Reduced pain and improved function

Clarke et al65

Randomized controlled trial (Level of evidence:1)

46 Lateral elbow tendinopathy Autologous skin-derived tendon-like cells

6 month Clinical, ultrasound

Improved function of the patients and ultrasonographical charactristics of the tendons based one the tendon thickness, hypoechogenicity, intra-substance tears, and neovascularity

Silva et al84

Randomized controlled trial (Level of evidence:2)

43 ACL injury and reconstruction Adult non-cultivated bone marrow stem cells

3 month MRI Was not effective in bone tunnel healing

Qu et al90 Randomized controlled trial

72 Bone nonunion fracture of femur and tibia

MSCs transplantation derived from human umbilical cord

13.2 month Clinical tests Stem cell therapy increased bone union healing

40

Figure captions

Fig. 1.

Mesenchymal stem cells can be obtained from different sources. The most reliable sources for harvesting MSCs are

bone marrow, adipose tissue, circulating blood and cord blood. After cell extraction, stem cells could be directly

injected into the tissue so that the growth factors and cytokines present in the tissue can differentiate them into the

tissue specific cells. Differentiation also can be done in laboratory. In the tissue, the differentiated cells are going to

be matured. For example MSCs can be differentiated into the tenoblasts or osteoblasts and during healing coarse the

become mature and are changed into osteocytes and tenocytes, respectively. MSCs can be differentiated into several

different tissue specific cells such as adipose, tenoblasts, osteoblast, chondroblast, etc.

Fig. 2.

Embryonic and fetal stem cells are other source of pluripotent stem. They can be differentiated into several different

tissue specific cells. Embryonic stem cells are more famous than fetal stem cells and the fetal stem cells have lower

potency than the embryonic cells. Application of these types of stem cells is faced with much ethical concerns

especially in human medicine. However their application in veterinary medicine is done by many researchers.

Embryonic stem cells are mostly derived from inner cell mass of the blastocyst and fetal stem cells can be obtained

from different tissues of the fetus however, they are more tissue specific and more differentiated than embryonic

stem cells.