Mesenchymal stromal cells for cartilage repair in osteoarthritis

Accepted Manuscript Mesenchymal stromal cells for cartilage repair in Osteoarthritis Murali Krishna Mamidi, Anjan Kumar Das, Zubaidah Zakaria, Ramesh Bhonde PII:

S1063-4584(16)01060-8

DOI:

10.1016/j.joca.2016.03.003

Reference:

YJOCA 3715

To appear in:

Osteoarthritis and Cartilage

Received Date: 15 May 2015 Revised Date:

9 February 2016

Accepted Date: 3 March 2016

Please cite this article as: Mamidi MK, Das AK, Zakaria Z, Bhonde R, Mesenchymal stromal cells for cartilage repair in Osteoarthritis, Osteoarthritis and Cartilage (2016), doi: 10.1016/j.joca.2016.03.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Mesenchymal stromal cells for cartilage repair in Osteoarthritis

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Murali Krishna Mamidi1, Anjan Kumar Das2, Zubaidah Zakaria3, Ramesh Bhonde1¶

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School of Regenerative Medicine, Manipal University, Bangalore - 560065, India.

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Department of Surgery, Taylor’s University School of Medicine, Sungai Buloh Hospital,

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Selangor, Malaysia.

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50588 Kuala Lumpur, Malaysia.

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Hematology Unit, Cancer Research Centre, Institute for Medical Research, Jalan Pahang,

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Corresponding author (e-mail: [email protected]; fax: +91 80 24860691)

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Keywords: Mesenchymal stem cells, regeneration, cell therapy, clinical trials,

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osteoarthritis.

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Running title: Cell therapy for OA.

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ACCEPTED MANUSCRIPT Abstract

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Treatment for articular cartilage damage is quite challenging as it shows limited repair and

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regeneration following injury. Non-operative and classical surgical techniques are

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inefficient in restoring normal anatomy and function of cartilage in osteoarthritis (OA).

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Thus, investigating new and effective strategies for OA are necessary to establish feasible

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therapeutic solutions. The emergence of the new discipline of regenerative medicine,

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having cell-based therapy as its primary focus, may enable us to achieve repair and restore

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the damaged articular cartilage. This review describes progress and development of

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employing mesenchymal stromal cell (MSC)-based therapy as a promising alternative for

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OA treatment. The objective of this review is to first, discusses how in vitro MSC

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chondrogenic differentiation mimics in vivo embryonic cartilage development, secondly, to

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describe various chondrogenic differentiation strategies followed by pre-clinical and

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clinical studies demonstrating their feasibility and efficacy. However, several challenges

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need to be tackled before this research can be translated to the clinics. In particular, better

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understanding of the post-transplanted cell behaviour and learning to enhance their potency

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in the disease microenvironment is essential. Final objective is to underscore the

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importance on isolation, storage, cell shipment, route of administration, optimum dosage

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and control batch to batch variations to realise the full potential of MSCs in OA clinical

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trials.

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ACCEPTED MANUSCRIPT INTRODUCTION

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Osteoarthritis (OA) is a “wear-and-tear” kind of disease which ultimately results in

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degeneration of articular cartilage. Knees, hips, joints in the hands and spine are the most

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commonly affected. The surface layer of the articular cartilage breaks down and wears

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away which causes rubbing of bones and leads to pain, swelling and stiffness. OA has the

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4th greatest impact on worldwide health by 2020, considering death and disability and is

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the fastest growing major health condition1.

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Epidemiology: OA is estimated to affect 250 million people worldwide. It mainly impacts

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older individuals; knee osteoarthritis in men aged 60 to 64 are commoner in the right side

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(23% right knee; 16.3% left knee), while this distribution is balanced in women of the

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same age2,3 (24.2% of right knee; 24.7% of left knee). A variety of endogenous (e.g., age,

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sex) and exogenous (obesity, patient’s lifestyle) risk factors for OA have also been

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outlined4. Recently, a number of genome wide association studies (GWAS) highlighted

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the significance of gene mutations (e.g., in GDF5) for the development of knee OA5,6.

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Construction workers have a significantly higher prevalence of knee OA2 highlighting the

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importance of wear and tear in the pathogenesis of OA. Disease onset is gradual, usually

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begins after the age of 40 and women are more prone to OA compared to men1. Damage is

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maximum in load bearing joint such as knee. Thus, weight loss is a preventive measure to

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avoid OA.

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OA- Yesterday, Today and Tomorrow: OA was viewed as an inevitable consequence of

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aging or injury, about which little could be done. Patients were told to rest their joints by

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avoiding exercise. For pain relief, patients took aspirin or, beginning in the mid-1970s,

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nonsteroidal anti-inflammatory drugs (NSAIDs) such as ibuprofen. Artificial knees which

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were developed to replace damaged joints were constructed like hinges that did not permit

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ACCEPTED MANUSCRIPT natural rotation or bending of the knees. As a result, many implants loosened shortly after

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surgery. In 2006, approximately 542,000 total knee replacement surgeries were performed

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in the United States (NIH Fact Sheets; Osteoarthritis). While conventional treatments like

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physiotherapy or drugs offer temporary relief of clinical symptoms, restoration of normal

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cartilage function has been difficult to achieve. Moreover, in severe cases of knee

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osteoarthritis total knee replacement may be required. Total knee replacements come

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together with high effort and costs and are not always successful. As of now there is no

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radical treatment available to cure OA. Hence, there is a dire need to develop new and

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pragmatic treatment modalities to combat OA enhancing their repair efficiency in

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biological way.

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Cartilage repair and regeneration: Can it be achieved by cell-based therapies via

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mesenchymal stromal cells?

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Cell-based therapies have been shown to reverse the symptoms and pathophysiology of

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OA7. With this in mind, autologous cultured chondrocyte transplantation for cartilage

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regeneration has been used successfully for over a decade8. This method requires cartilage

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biopsy samples and donor site derived chondrocytes have been shown to de-differentiate

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during expansion with limited life span following transplantation9. These difficulties left

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the field open to other therapies and the most promising is the use of mesenchymal stromal

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cells (MSCs) to repair the damaged cartilage tissue. The objective of using MSCs is to

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support the process of cartilage repair and regeneration within the knee joint10. Even

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synovial fluid inside the joint contains MSCs which can differentiate into chondrocytes.

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However, the number of endogenous MSCs available for chondrocyte differentiation is

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limited11. Therefore, external MSC transplantation might improve the joint cartilage repair

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and regeneration.

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ACCEPTED MANUSCRIPT In the recent past, MSCs were identified in all organs and tissues of adult mice except the

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peripheral blood12. Also human MSCs have been isolated from several tissues including

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peripheral blood13, 14. Regardless of their origin they have the capacity to differentiate into

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many cell types depending on the stimulus15. One of the important properties of MSCs is to

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retain immunomodulatory activity due to lack of human leucocyte antigen (HLA) class II

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expression16,

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molecules which are required for alloreactive T-cell activation18, 19, 20 making them fit for

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allogeneic transplantation. Furthermore, differentiated phenotypes of MSCs such as,

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chondrocytes, adipocytes and osteocytes have also been shown to be non-immunogenic in

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nature21, 22. Collectively, these results suggest that the allogeneic MSCs transplantation can

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be accomplished without HLA matching.

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MSCs are known to secrete a large number of growth factors, cytokines, and chemokines

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for mediating various functions including anti-inflammatory, anti-apoptotic, anti-fibrotic,

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angiogenic, mitogenic, and wound-healing through paracrine activity23. All these features

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of MSCs are highly desired and support their candidature for therapeutic purposes.

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In vitro chondrogenic differentiation of MSCs mimics in vivo embryonic cartilage

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development:

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Mounting evidence suggests that the in vitro MSC chondrogenic differentiation mimics in

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vivo embryonic chondrogenic differentiation24 (Fig. 1). In vitro MSC expansion phase may

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correspond to the initial proliferation of mesenchymal cells before condensation and

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switching over to the high-density MSC pellet cultures may mimic the in vivo

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condensation step of chondrogenesis during embryonic development. Biomechanical

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forces employed on the tissue during radial expansion result in the formation of highly

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heterogeneous tissues. The outer layer encompasses flattened, undifferentiated MSCs and

. Moreover, MSCs have been shown to be negative for co-stimulatory

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ACCEPTED MANUSCRIPT chondrocyte-like morphology is observed beneath the outer layer enclosed within ECM

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molecules and the inner core is poorly differentiated25.

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Chondrogenic differentiation strategies:

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Exogenous inductive molecules: To achieve chondrogenic differentiation the most

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essential is to employ well characterized soluble factors. These including transforming

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growth factor (TGF)-β which is a multifunctional peptide that controls proliferation and

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differentiation of many cell types, bone morphogenetic protein (BMP) plays an important

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role in early skeletal development, and insulin growth factor (IGF)-1 controls cell

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proliferation and differentiation26,

. The effect of the growth factors (GFs) differs

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depending on the dose, duration of treatment and cell development/differentiation stage.

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MSC chondrogenic differentiation with these GFs requires repeated treatments at high

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concentrations, expensive and may cause side effects28. Despite these disadvantages well

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characterized GFs are essential and future research demands the testing of small molecules,

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pharmaceuticals and naturaceuticals to achieve enhanced MSC chondrogenic potential.

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Co-culture techniques: Enhanced chondrogenic phenotypes were observed during co-

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culture of MSCs with CD45 positive cells39. Co-culture of MSCs with juvenile articular

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chondrocytes resulted in efficient chondrogenic differentiation30. It has been observed that

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co-culture with primary chondrocytes and mechanical stimulation helped to achieve MSC

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chondrogenic differentiation without biochemical agents31. Co-cultures of MSCs and

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articular chondrocytes (ACs) with reduced concentration and duration of TGF-β3 showed

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effective chondrogenic potential32,

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technique for MSC cartilage engineering.

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Cell to cell interactions/cross-talk: MSC three-dimensional (3D) cultures showed better

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chondrogenic potential because of enhanced cellular interactions when compared to

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. These studies highlight the promise of co-culture

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ACCEPTED MANUSCRIPT monolayer cultures34. Neighbouring cells and the cell-adhesion molecules enable MSCs to

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differentiate into prechondroblasts by modulating the local microenvironment because of

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strong cellular interaction34. It has been shown that the maintenance of appropriate cell

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density is crucial to achieve required cell-to-cell interactions35. MSCs pellet cultures

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showed enhanced cartilage specific gene expression profile because of strong cell-cell

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interactions36. MSC 3D cultures demonstrated elevated production of collagen type II,

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aggrecan, and cartilage oligomeric matrix protein (COMP) when compared to MSC

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monolayer cultures37. Recent work suggested that the cellular interactions can be regulated

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by the matrix composition thereby controlling the stem cell chondrogenic fate38. Together,

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these studies highlight the importance of cellular interactions and cross-talk to achieve

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better MSC chondrogenesis. These studies enforce strategic planning in getting maximum

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chondrogenic differentiation in vitro and later on to apply these modalities for in vivo

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cartilage repair.

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Three-dimensional niche creation using scaffolds: Hyaluronic acid (HA) is a natural

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matrix provides a stable three-dimensional environment for MSC chondrogenesis39. HA

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hydrogel facilitates diffusion of cells and nutrients which eventually increases ECM and

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chondrocyte synthesis40. Synthetic scaffolds such as poly L-lactide-ε-caprolactone have

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been shown to produce similar structures to the natural cartilage41. Hybrid scaffolds

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composed of Poly (N-isopropylacrylamide) (PNIPAAm) and water soluble chitosan

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(WSC) showed enhanced MSC chondrogenesis42. It has been shown that the alginate (Alg)

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foam scaffold supplemented CS resulted in better MSC chondrogenic phenotypes43. Poly

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(L-lactic acid) (PLLA) and chitosan (CHT) scaffolds have been showed to maintain a

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hyaline-like phenotype of MSCs and prevented the progression of hypertrophic process44.

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Combining MSC-based cell therapies with biomaterials lead to significant improvement in

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repair and regeneration of OA joints45. These studies demonstrate the significance of the

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ACCEPTED MANUSCRIPT scaffolds on the cellular events of chondrogenesis and direct towards injecting scaffolds

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loaded with MSCs for better in vivo results.

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Additional methods: MSCs were also tested for chondrogenic potential by subjecting

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them to hypoxic culture conditions, preconditioning with suitable adjuvants and genetic

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manipulation to achieve better chondrogenenis. The idea is to inject MSCs after

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preconditioning to get high efficiency cartilage repair within short span of time. All these

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approaches cumulatively suggests that the MSCs are the suitable cell type for treatment of

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OA and this was further tested in small, large animals including humans was discussed

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below.

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MSCs tested for OA in pre-clinical models

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Small animal models: Studies explored the use of MSCs in OA disease models showed

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improved cartilage repair. Transplantation of MSCs encapsulated in self-assembled peptide

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(SAP) hydrogels showed chondroprotection and reduced subchondral bone mineral density

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in a rat OA model46. Intra-articular injection of atsttrin (TNFα blocker) transduced MSCs

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in an OA animal model showed improvement by suppression of matrix proteases and

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inflammatory factors47. Chondro protection was observed when MSCs injected into rat

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knee joints after a hemi-meniscectomy48. MSCs administered intra-articularly into

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damaged joints resulted in inhibition of articular cartilage degeneration in the leporine

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models49, 50, 51. These results were further tested in large animal models.

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Large animal models: Intra-articular injection of MSCs in dog OA model showed

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improvement with time52. The histological and macroscopic findings showed significant

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improvement of meniscal repair with MSCs in a porcine model53. Intraarticular injections

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of MSCs resulted meniscus regeneration and provide protection at the medial femoral

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articular cartilage in a porcine meniscal defect model54. Horses with femorotibial lesions

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ACCEPTED MANUSCRIPT when subjected to combine treatment of surgery and intra-articular MSC administration

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showed improvements55. Although pre-clinical studies proves the safety and efficacy of

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MSCs for OA but few issues discussed below requires attention to resolve before cell

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therapy reaches clinics.

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Major challenges to be addressed

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Better understanding about post-transplanted MSC behaviour: Cell engraftment

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studies revealed that small proportion of MSCs home and retain in the target sites. Despite

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MSCs vanishing in short time periods, improvement of the disease condition was observed

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without significant numbers of cells reaching the target organs56. In these settings, it is

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likely that the recovery may not be because of cellular regeneration but rather due to the

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secreted/stimulated biomolecules by MSCs. If it turns out to be true, then one needs to

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think about how long such stimulus should persist inside the body and whether the stem

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cell secreted/stimulated biomolecules can be characterized and administrated as drugs.

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Cumulatively, these results suggest that the complete potential of MSCs have not yet been

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exploited.

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How can we enhance the potency of the transplanted MSCs into the disease

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microenvironment? MSCs have been used in OA animal models and clinical studies

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(refer below) to investigate their capacity to repair the damage. The preclinical/clinical

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outcome was often encouraging but understanding how stem cells respond to the hostile

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disease microenvironment remains unknown. Also shifting the cells from in vitro normoxic

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culture conditions to in vivo hypoxic niche might affect the survival of MSCs after

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transplantation. For these reasons, it is possible that the action of MSCs might be further

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improved by several protocols such as genetic manipulation, hypoxic treatment etc., to

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reinforce their potency and thus enhance the healing processes in damaged tissue following

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ACCEPTED MANUSCRIPT transplantation. Further, we anticipate that the physical, physiological and pharmaceutical

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preconditioning of MSCs might enable their post-transplantation potency and efficacy.

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Current clinical approaches employing MSCs for OA management:

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Clinical trials: The results of clinical studies have been widely variant. This is not

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unexpected if one looks at the possible problems of conducting a clinical trial. It is best not

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to speculate immediately on the possibility of MSC efficacy in OA of the knee. Several

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clinical trials are on which will probably give us some answers (Table-1). There have been

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several recent reviews which have summarised the present clinical evidence on this subject

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to which the reader is referred57, 58. This review concentrates on the problems faced by

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investigators conducting a trial to assess efficacy of MSCs in OA.

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Conduction of a good quality clinical trial for OA is fraught with hazards. In our

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experience59 the problem of accurately evaluating the progression of the disease is an

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important issue. Various scoring systems do exist, and are used widely, but the

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proliferation of scoring systems and debates about their relative merit suggest that they

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may not always be an accurate assessment of the disease progression. In any case, the

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divergent scoring systems make it difficult to compare across studies. In addition, the

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evaluation of cartilage regeneration is also extremely difficult. The whole-organ magnetic

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resonance imaging score (WORMS) scoring system is the most widely used, but leaves

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much to be desired. The scoring is difficult and presupposes extensive training before it

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can be evaluated. One other unanswered question is when we can expect cartilage growth

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which can be evaluated radiologically? In 6 months (duration of follow up for most

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studies), 12 months or longer? The question of adding a natural or a synthetic scaffold to

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ensure cell migration to all parts of the joint and the attachment of the cell to the

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ACCEPTED MANUSCRIPT appropriate tissue has been debated. Most research indicates that using a natural scaffold

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like hyaluronic acid is useful, though hard evidence is lacking.

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Blinding the product can also be difficult. The cells are usually dispensed in syringes

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which are covered to blind the investigator to the contents. However the volume of the

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injected material has to be limited to 2-4 ml ideally. Cells tend to clump at the bottom of

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the syringe and the syringe must be vigorously rotated in the palms of the hands in order

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that the cells be suspended uniformly. This process can potentially un-blind the study as

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the injected material is clear for the placebo group while it is cloudy for the cell group. The

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viscosity of the injection is also different and easily discernible to the investigator. This

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can vitiate the results of an ostensibly blinded trial.

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Problems associated with clinical translation

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The path from the laboratory to the bedside is strewn with thorns. Many obstacles need to

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be crossed in order to have a successful transplant strategy. These include many potential

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issues, like the problem of transporting the cells to the end user without loss of potency and

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numbers. Another question that arises is about the transplant strategy. What should the

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transplant strategy be? Should the cells be delivered systemically or locally; and if locally,

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by injection or by a more invasive surgical procedure? There are several other questions

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that have exercised groups which have tried to translate the in vitro work to the patient.

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Many of these issues need research and solutions.

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Manufacture of MSCs: Growing MSCs in the lab is easier than the culture of embryonic

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and induced pluripotent stem cells (iPSCs) which require baby sitting in almost the literal

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sense. However, when culture of the MSCs is scaled up to industrial levels to provide the

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numbers required for clinical/therapeutic applications problems emerge. While

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manipulating the cells in vitro to produce large numbers is fairly straight forward the

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ACCEPTED MANUSCRIPT bigger challenge lies in ensuring homogeneous population. How similar are these cells in

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terms of their identity, molecular signature, gene expression patterns, differentiation

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potential and phenotype? Although there are international guidelines in place to identify

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MSCs there is a lot of ambiguity. MSCs are most often identified by a battery of surface

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markers60 which are shared with other cell types. Hence, there is always difficulty in

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ensuring a pure culture of MSCs in the strictest sense.

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The second area of concern is the manufacturing of MSCs for clinical use or clinically

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eligible MSCs. To make cells clinically eligible they need to be isolated in xenofree

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conditions followed up scaling them to create numbers required for transplantation. This

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typically requires the culture conditions devoid of fetal calf/bovine serum, animal feeders

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or extracellular matrices like fibronectin/laminin/matrigel/poly-ornithine or combinations

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of them. It is also essential to replace the enzymes like trypsin, dispase and collagenase etc.

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from non-animal61 sources or utilize mechanical methods.

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Problems with storage: The primary advantage of MSCs is the ability to use cells from

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allogeneic sources. Hence, it is imperative to bank them and make them available as off the

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shelf product. There have been numerous attempts to cryopreserve MSCs. However these

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methods are fraught with risk. The key to long term viability is the type of

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cryopreservation medium used. Scientists have suggested and tested various combinations

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and materials to this effect62. However, most freezing mixtures utilise DMSO which is

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known to be toxic to certain organs of the body at specified concentrations63. As a result, it

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is essential to wash away or dilute the DMSO prior to administration. This essential step in

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the delivery and dispensing of MSCs limits widespread application.

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Secondly, post thaw viability and functionality of the MSCs is another point of concern.

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This also depends upon whether the cells have been handled and freeze-thawed with best

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ACCEPTED MANUSCRIPT practice standards. It thus becomes people specific and small deviations in handling or

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protocol can have drastic effects on MSC viability and functionality. If not freeze-thawed

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appropriately, the viability can be down to less than 50% which is not optimal for the

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patient and is unlikely to have any positive consequence on the disease.

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Thirdly, a lot of speculation still revolves around how long these cells can be stored in

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vapour phase of liquid nitrogen. Data do suggest that even after 5 years after storage the

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post thaw viability may be well above the permissible limit64. This needs further

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validation. Companies working in this space claim to have solved some of these problems,

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but in the absence of openly available data, the question must be left open. Recent

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evidence from Sharma and Bhonde suggests that cryopreservation of MSCs for more than

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six months leads to genetic instability and decreased cell proliferation65.

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Fourthly, transportation of MSCs to the site of administration is a logistic issue worthy of

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mention. MSCs stored in liquid nitrogen need to be transported at the same temperature

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which is practically not a viable option. This restricts the application of MSCs to the

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location where they are stored. It is possible to transport cells in so called “cold boxes”

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which maintain the temperature at about 2-8°C. This can maintain the viability of the cells

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for about 6 hours55. The cells must be transplanted within this time. This can limit the use

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of therapy to only specific areas and hospitals with specialised equipment and defeats the

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purpose of developing an easily used therapy.

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Quality control issues

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Are all batches bioequivalent? Pharmacologically "two pharmaceutical products are

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bioequivalent if they are pharmaceutically equivalent and their bio-availabilities (rate and

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extent of availability) after administration in the same molar dose are similar to such a

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degree that their effects, with respect to both efficacy and safety, can be expected to be

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ACCEPTED MANUSCRIPT essentially the same66. While this is easily established in chemical pharamacology, things

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are not so straight forward for cells or any biologically active ingredient. The activity of

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cells would depend on many individual steps: the donor health, the tissue collection

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technique, transportation and in vitro manipulations in the lab. Although most of these can

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be tackled with supervision and care, we do not have control over donor health. Donors are

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screened for most obvious diseases; family history is recorded to understand genetic

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predispositions if any, lifestyle/habits/diet and occupation are noted to understand the

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potential exposure to any carcinogens/mutagens etc. But as we all would agree, no two

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individuals are the same and hence their cells cannot be the same.

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To achieve some semblance of order in this randomness, it is possible to pool or mix the

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cells from several different donors. It is however essential to bear in mind that there would

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be intra and inter donor pool variations. Amongst the pool, some donors would

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outperform/underperform the others based on their growth kinetics, differentiation

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potential and genetic predisposition. Hence, attaining bioequivalence is not possible with

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the current parameters used for measuring the biopotential of these cells. We need to

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identify more markers specific to cell functioning to be able to measure and understand

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whether they would be bioequivalent or not. Accordingly, all similar donors can be pooled

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in one batch for manufacturing.

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Routes of administration: Most if not all clinical attempts to treat OA of the knee have

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used local administration of the cells to the joint as opposed to the systemic route. This is

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logical as the principal cause of the disease is a local degeneration of the joint mechanism

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because of abnormal stress. Examination of the joint fluids in such patients also suggests

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that proinflammatory markers may be present67. These findings suggest that the symptoms

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of OA are the result of local factors which could include pathologies related to the

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cartilage, synovium and other joint structures. It appears logical therefore that the cells

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ACCEPTED MANUSCRIPT should be locally administered and most groups have used this approach, either by joint

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injection or by an arthroscopic approach68, 69, 70.

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However proper needle placement in the joint needs more attention that it has been

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accorded in the past. Studies have shown that in one third of cases; even experienced

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clinicians may be unable to enter the joint successfully. It is advisable to use

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ultrasonographic or fluoroscopic guidance during the procedure71. Most published studies

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do not appear to have heeded this advice. Another important issue that must be addressed

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when writing the protocol is that intra articular injection has a marked placebo effect72.

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This effect which can vitiate the results of clinical trials may explain widely varying results

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obtained in open label studies as opposed to randomised trials.

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Future Direction

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It is reasonable to expect that MSCs will prove to be an important therapy for OA (Fig. 2).

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These cells have many advantages which suggest that they may be the ideal solution to the

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OA problem. They are easily cultured, can be expanded, have anti-inflammatory and

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immune-modulatory properties, can be used from allogeneic sources and are expected to

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regenerate cartilage. However the actual clinical translation has taken much longer than

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one would have expected in the early years of cell therapy. These problems are related to

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culture, storage and transport to the clinic and the conduct of good quality clinical trials.

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Some of these problems have been elaborated above. Research now needs to be directed at

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resolving these problems (Table-2). It is entirely possible that solutions to these problems

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will bring about the era of cell therapy for OA, a goal which has been the hope of all those

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who work with this patient group. Therapeutic usage of MSCs may provide regenerative

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medicine for degenerative cartilage and improve upon the quality of elderly life.

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ACCEPTED MANUSCRIPT Acknowledgements: The authors thank Manipal University, Manipal, India, and Taylor’s

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University School of Medicine, Selangor Malaysia for supporting this study. The authors

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wish to thank the Director General of Health, Malaysia for his permission to publish this

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paper. Acknowledgements are also due to Dr. Rajarshi Pal & Vijaya Bhaskar Reddy

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Konala for checking the manuscript and helping in drawing figure-1 respectively.

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References:

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Current evidence on risk factors for knee osteoarthritis in older adults: a systematic

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review and meta-analysis. Osteoarthritis Cartilage 2015; 23(4):507-515.

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ACCEPTED MANUSCRIPT Author contributions:

2

MKM drafted introduction, in-vitro chondrogenic differentiation of MSCs mimics in vivo

3

embryonic cartilage development, chondrogenic differentiation strategies and the

4

preclinical sections of this manuscript. AKD drafted clinical section of this manuscript

5

along with problems associated with clinical translation and future direction. ZZ

6

coordinated for drafting of this manuscript. RRB conceived idea, design of this manuscript

7

and coordinated for drafting of this manuscript with assistance as noted above. All authors

8

read and approved the final manuscript.

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Conflict of Interest: The authors declare that there is no conflict of interest.

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Figure Legends:

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Figure-1: Stages of in vivo embryonic cartilage development compared with in vitro MSC

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chondrogenic differentiation.

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Figure-2: Road map of MSCs for cartilage repair in OA.

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ACCEPTED MANUSCRIPT 1

Tables:

2

Table-1: Stem cells based clinical trials for articular cartilage defects for the treatment of

3

OA in different countries. Title (Interventions/indication)

Mesoblast, Ltd.

Brazil

Paulo Brofman

Canada

Jas Chahal

China

Cellular Biomedicine Group Ltd

Safety and Efficacy Study of MSB-CAR001 in Subjects 6 Weeks Post an Anterior Cruciate Ligament Reconstruction Autologous Bone Marrow Mesenchymal Stem Cells Transplantation for Articular Cartilage Defects Repair Human Autologous MSCs for the Treatment of Mid to Late Stage Knee OA Autologous Adipose Tissue Derived Mesenchymal Progenitor Cells Therapy for Patients With Knee Osteoarthritis Autologous Adipose TissueDerived MPCs Therapy for Knee OA UC-MSCs Transplantation in Articular Cartilage Defect

TE D

EP

Cellular Biomedicine Group Ltd Shenzhen Hornetcorn Biotechnology Company, LTD Cairo University School of Medicine University of Marseille

AC C

Egypt

France

University Hospital, Montpellier; Nantes University Hospital; University

Phase-1 Phase-2

Phase-1 Phase-2

M AN U

Australia

Trail stage

The Use of Autologous BMMSCs in the treatment of articular cartilage defects Transplantation of BM-MSCs stimulated by Proteins Scaffold to Heal Defects Articular Cartilage of the Knee Autologous adipose derived stem cells administrated for intra-articular use (ADIPOA) Immunomodulatory Adult MSCs for chondrogenic potential (ARTHROSTEM) Osseous setting improvement 27

ClinicalTrials.gov Identifier (Estimated enrolment) NCT01088191 (24)

RI PT

Sponsor

SC

Country

NCT01895413 (10)

Phase-1 Phase-2

NCT02351011 (12)

Phase-1 Phase-2

NCT01809769 (18)

Phase-2

NCT02162693 (48)

Phase-1

NCT02291926 (20)

Phase-2 Phase-3

NCT00891501 (25)

Phase 0

NCT01159899 (50)

Phase-1

NCT01585857 (18)

Observat ional

NCT01879046 (30)

Phase-2

NCT00557635 (50)

ACCEPTED MANUSCRIPT

India

International Stem cell Services Limited Aditya K Aggarwal, Postgraduate Institute of Medical Education and Research Stempeutics Research Pvt Ltd

Allogeneic MSCs with Plasmalyte-A in OA

Royan Institute

MSC Transplantation in OA of Hip Joint Royan Institute Side Effects of Autologous MSCs Transplantation in Ankle Joint OA Royan Institute Autologous Transplantation of MSCs and Scaffold in Full-thickness Articular Cartilage Royan Institute The Effects of Intra-articular Injection of MSCs in Knee Joint OA Royan Institute Transplantation of BM-MSCs in Knee OA by Rheumatoid Arthritis Royan Institute Articular Cartilage Resurfacing With MSCs in OA of Knee Joint Tehran University stem cell transplantation for of Medical Treatment of Knee OA Sciences University of Mesenchymal Stem Cells in Jordan Knee Cartilage Injuries K-Stemcell Co Autologous Adipose Tissue Ltd Derived Mesenchymal Stem Cells Transplantation in Patient With Degenerative

AC C

EP

TE D

Iran

Safety and Efficacy of Autologous Bone Marrow Stem Cells for Treating Osteoarthritis MSCs Enhanced With PRP Versus PRP In OA Knee (MSCPRPOAK)

Jordan Korea

Observat i-onal

NCT01038596 (30)

RI PT

University Hospital Dresden

Phase-1 Phase-2

NCT01152125 (10)

Phase-1 Phase-2

NCT01985633 (24)

SC

Germany

with Co-implantation of Osseous Matrix and Mesenchymal progenitors cells from autologous Bone Marrow MSCs for OA

M AN U

Hospital, Clermont-Ferrand

28

Phase-2

NCT01453738 (60)

Phase-1

NCT01499056 (6)

Phase-1

NCT01436058 (6)

Phase-1

NCT00850187 (6)

Phase-2

NCT01504464 (40)

Phase-2 Phase-3

NCT01873625 (60)

Phase-1

NCT01207661 (6)

Phase-1

NCT00550524 (5)

Phase-2

NCT02118519 (12)

Phase-1 Phase-2

NCT01300598 (18)

ACCEPTED MANUSCRIPT

Mexico

NCT01626677 (103)

Phase-2

NCT01448434 (72)

SC

RI PT

Phase-3

NCT01459640 (50)

Phase-2

Autologous Stem Cells in OA Phase-1

NCT01485198 (30)

Ullevaal University Hospital

MSCs for articular cartilage defects

Phase-1

NCT00885729 (50)

MSCs for articular Cartilage Defects

Phase-1

NCT00885729 (50)

University Hospital of North Norway

ACI-C Versus AMIC. A Randomized Trial Comparing Two Methods for Repair of Cartilage Defects in the Knee Safety and Feasibility of a Single-stage Procedure for Focal Cartilage Lesions of the Knee. Safety and Feasibility Study of Autologous Stromal Vascular Fraction (SVF) Cells for Treatment of Osteoarthritis Clinical Study of UC-MSC for Treatment of OA Safety and Feasibility Study of Mesenchymal Trophic Factor (MTF) for Treatment of OA

Phase-2 Phase-3

NCT01458782 (80)

Phase-1 Phase-2

NCT02037204 (35)

Phase-1 Phase-2

NCT01885832 (20)

Phase-1 Phase-2 Phase-1 Phase-2

NCT02237846 (40)

EP

Oslo University Hospital

UMC Utrecht

AC C

Netherlan ds

NCT01041001 (104)

Hospital Universitario Dr. Jose E. Gonzalez

TE D

Norway

Phase-3

M AN U

Malaysia

Arthritis Medipost Co., Ltd Compare the Efficacy and Safety of Cartistem® and Microfracture in Patients With Knee Articular Cartilage Injury or Defect Medipost Co Ltd. Follow-Up Study of CARTISTEM® Versus Microfracture for the Treatment of Knee Articular Cartilage Injury or Defect Stempeutics Allogeneic MSCs with Research Plasmalyte-A in OA Malaysia SDN BHD NUM & Intra-Articular Autologous Cytopeutics Pte. BM-MSC Transplantation to Ltd Treat Mild to Moderate OA

Translational Biosciences

Panama Translational Biosciences Translational Biosciences

29

NCT02003131 (40)

ACCEPTED MANUSCRIPT

Russia

Burnasyan Federal Medical Biophysical Center

United States

Institute of Regenerative and Cellular Medicine; Ageless Regenerative Institute;

Treatment of Knee OA with Allogeneic MSCs ASC Therapy for Repairing Articular Cartilage in Gonarthrosis Effectiveness and Safety of Autologous ADRC for Treatment of Degenerative Damage of Knee Articular Cartilage Safety and Clinical Effectiveness of A3 SVF in Osteoarthritis

Phase-1 Phase-2

NCT01399749 (30)

Phase-1 Phase-2

NCT02123368 (30)

Phase-1 Phase-2 Phase-1 Phase-2 Phase-1 Phase-2

NCT01183728 (12)

Phase-1 Phase-2

NCT02219113 (12)

RI PT

Clinica Universidad de Navarra Red de Terapia Celular Red de Terapia Celular Banc de Sang i Teixits

Autologous MSCs vs. Chondrocytes for the repair of chondral knee defects (ASCROD) Treatment of Knee OA by Intra-articular Injection of BM-MSCs Autologous BM-MSCs

SC

La Paz University Hospital

M AN U

Spain

AC C

EP

TE D

Autologous Adipose-Derived Stromal Cells Delivered Intra-articularly in Patients With Osteoarthritis Mayo Clinic; Use of Autologous Bone Marrow Aspirate Concentrate in Painful Knee Osteoarthritis (BMAC) Medipost Co Ltd; Evaluation of Safety and Exploratory Efficacy of CARTISTEM®, a Cell Therapy Product for Articular Cartilage Defects University of Autologous Adipose Stem Science Ho Chi Cells and Platelet Rich Minh City Plasma Therapy for Patients With Knee Osteoarthritis StemGenex Outcomes Data of Adipose Stem Cells to Treat Osteoarthritis Regenerative Pain Outcomes Data of Bone Center, Illinois Marrow Stem Cells to Treat Hip and Knee Osteoarthritis Mayo Clinic Use of Autologous Bone Marrow Aspirate Concentrate in Painful Knee Osteoarthritis (BMAC) 30

NCT01586312 (30) NCT01227694 (15)

Not provided

NCT01947348 (30)

Phase-1 Phase-2

NCT01739504 (500)

Phase-1

NCT01931007 (25)

Phase-1 Phase-2

NCT01733186 (12)

Phase-1 Phase-2

NCT02142842 (32)

Not provided

NCT02241408 (50)

Not provided

NCT01601951 (12)

Phase-1

NCT01931007 (25)

ACCEPTED MANUSCRIPT

Arizona Pain Specialists

Not provided

NCT01978639 (300)

Not provided

NCT01953523 (3000)

Table -2: Major clinical and research focus for clinical translation of MSCs for OA. Clinical focus Storage of MSCs

Research focus

Behaviour of post-transplanted MSC How to enhance potency of the transplanted MSCs Characterization of MSC

How to deliver cells?

Classification according to differentiation potential

TE D

Transport of MSC to the clinical trial site Blinding the investigator

EP

How to estimate clinical progress?

AC C

How to evaluate radiological improvement? Standardized procedures

3

NCT01908777 (33)

M AN U

1 2

Phase-2

SC

Elliot Lander

Multicenter Study of High Dose Chemotherapy With Autologous Stem Cell Transplant Followed by Maintenance Therapy With Romidepsin for the Treatment of T Cell Non-Hodgkin Lymphoma Injections of FloGraft Therapy, Autologous Stem Cells, or Platelet Rich Plasma for the Treatment of Degenerative Joint Pain Safety and Clinical Outcomes Study: SVF Deployment for Orthopedic, Neurologic, Urologic, and Cardiopulmonary Conditions

RI PT

Memorial Sloan Kettering Cancer Center

31

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT