Advancements in Regenerative Medicine

Austin Childress

Rotator cuff (RC) injuries are the most common cause of shoulder disability and pain in athletes and older adults, and the second most common musculoskeletal pathology behind lower back injury.^1,2 Partial and full thickness tears to the RC are associated with pain and instability and if left untreated often result in osteoarthritis and/or a complete loss of function. It is estimated that full thickness RC tears are found in 13% of individuals in their 50s, 25% in their 60s, and 50% in their 80s.³

Surgical repair is often indicated for RC tears with complete healing of the tendon ranging from 1 – 2.5 years.^2,4  A repaired tendon is less organized with an increased scar composition resulting in decreased overall mechanical strength and an increased probability of reinjury.⁴

The combination of tissue engineering with surgical repair has been suggested to stimulate stronger and more complete healing when compared to traditional methods.^4-9Tendon engineering therapies focus on Mesenchymal stem cells (MSC) due to their ability to differentiate into connective tissue.¹⁰

Engineered scaffolds are composed of biocompatible materials that support cell adhesion, cellular growth, the formation of three-dimensional tissue, and provide mechanical strength.^6-9Growth factors are involved in the activation and regulation of the cellular environmental response in tendon healing, and literature suggests that a 3D scaffold with sustained release of growth factors such as platelet-derived growth factor, transforming growth factor beta, vascular endothelial growth factor, insulin-like growth factor, and basic fibroblastic growth factor can provide the microenvironmental control needed to influence cell phenotype.⁶

Recent animal studies in rat and rabbit models have provided strong evidence suggesting that the use of MSCs with engineered scaffolds in conjunction with surgical repair improve the tendon to bone interface healing, collagen fiber alignment, increased rate of healing, and overall improved strength of the repaired tendon.^6-9,11 The literature offers compelling evidence to suggest that the placement of scaffolds seeded with MSCs into the tendon repair site during surgical repair of RC tears may provide improved overall recovery and a reduction in reinjury for patients.

1. Savin D, Meadows M, Verma N, Cole B. Rotator Cuff Healing: Improving Biology. Oper Tech Sports Med. 2017;25(1):34-40.
2. Depres-Tremblay G, Chevrier A, Snow M, Hurtig MB, Rodeo S, Buschmann MD. Rotator cuff repair: a review of surgical techniques, animal models, and new technologies under development. J Shoulder Elbow Surg. 2016;25(12):2078-2085.
3. Thomopoulos S, Parks WC, Rifkin DB, Derwin KA. Mechanisms of tendon injury and repair. J Orthop Res. 2015;33(6):832-839.
4. Saether EE, Chamberlain CS, Leiferman EM, et al. Enhanced medial collateral ligament healing using mesenchymal stem cells: dosage effects on cellular response and cytokine profile. Stem Cell Rev. 2014;10(1):86-96.
5. Adams SB, Jr., Thorpe MA, Parks BG, Aghazarian G, Allen E, Schon LC. Stem cell-bearing suture improves Achilles tendon healing in a rat model. Foot Ankle Int. 2014;35(3):293-299.
6. Govoni M, Berardi AC, Muscari C, et al. (*) An Engineered Multiphase Three-Dimensional Microenvironment to Ensure the Controlled Delivery of Cyclic Strain and Human Growth Differentiation Factor 5 for the Tenogenic Commitment of Human Bone Marrow Mesenchymal Stem Cells. Tissue Eng Part A. 2017;23(15-16):811-822.
7. Peach MS, Ramos DM, James R, et al. Engineered stem cell niche matrices for rotator cuff tendon regenerative engineering. PLoS One. 2017;12(4):e0174789.
8. Tornero-Esteban P, Hoyas JA, Villafuertes E, et al. Efficacy of supraspinatus tendon repair using mesenchymal stem cells along with a collagen I scaffold. J Orthop Surg Res. 2015;10:124.
9. Yokoya S, Mochizuki Y, Natsu K, Omae H, Nagata Y, Ochi M. Rotator cuff regeneration using a bioabsorbable material with bone marrow-derived mesenchymal stem cells in a rabbit model. Am J Sports Med. 2012;40(6):1259-1268.
10. Berebichez-Fridman R, Gomez-Garcia R, Granados-Montiel J, et al. The Holy Grail of Orthopedic Surgery: Mesenchymal Stem Cells-Their Current Uses and Potential Applications. Stem Cells Int. 2017;2017:2638305.
11. Sevivas N, Teixeira FG, Portugal R, et al. Mesenchymal Stem Cell Secretome Improves Tendon Cell Viability In Vitro and Tendon-Bone Healing In Vivo When a Tissue Engineering Strategy Is Used in a Rat Model of Chronic Massive Rotator Cuff Tear. Am J Sports Med. 2018;46(2):449-459.

Syed Zamin

Introduction. Osteoarthritis (OA) is a chronic condition due to the inflammation of synovium and breakdown of cartilage in joints which mainly affects the knees and hips^1-2. It has a lifetime prevalence of 40% in males and 47% in females and is steadily increasing due to increased life expectancy and a more sedentary lifestyle leading to increased obesity^3-4. The incidence of OA rises sharply after the age of 50 and generally peaks at the age of 70 for both sexes alike³. Studies have shown that altered chondrocyte homeostasis prevents the efficient repair of damaged cartilage, causing excess degradation through the production of matrix metalloproteases (MMPs)².

There are numerous ways to negate the effect of chondrocyte degradation and the progression of OA as found in literature. One method involves preventing large-scale bone reformation to decrease growth factor release by subchondral bone. Another method involves stem cell differentiation into chondrocytes. The latter method allows for the anti-inflammatory properties of mesenchymal stem cells (MSCs) to be coupled with the appropriate cell lineage differentiation and proliferation, restoring normal conditions to joints^5-6.

Methods. To demonstrate anti-inflammatory properties of MSCs, human bone marrow MSCs were suspended in complete culture medium (CCM) to form spheroids. The spheroid culture was then boiled and cooled to denature spheroid-secreted proteins, and the samples were assayed for PGE₂ using ELISA and for anti-inflammatory activity in macrophage assay⁵. To demonstrate chondrogenesis efficacy, bovine and rabbit MSCs were plated on scaffolds as part of co-cultures with articular chondrocytes (AC) at 1:1 or 1:3 AC-to-MSC ratios. Biopsies of the scaffolds were harvested at 0, 14, and 28 days⁶.

Results. ELISA studies of the spheroid cultures showed large amounts of PGE₂ present in solution. PGE₂ has been shown to have anti-inflammatory effects, as proven by abolishing the anti-inflammatory effects through an inhibitor of COX-2, through siRNAs for COX-2, and through antibodies for PGE₂⁵. All bovine and rabbit scaffold samples showed an increase in cellularity with bovine 1:1 co-cultures showing the greatest improvement. GAG synthesis was greatest in all of the co-cultures compared to pure AC cultures. The 28 day co-cultures showed gene expression levels for aggrecan and collagen types I and II to be greater than those of pure AC cultures⁶.

Conclusion. Chondrogenesis of MSCs can benefit patients suffering from OA in two manners. The anti-inflammatory effects of MSCs can reduce synovial inflammation and the chondrogenesis can help restore lost mass of cartilage.

1. Berenbaum F. Osteoarthritis as an inflammatory disease (osteoarthritis is not osteoarthrosis!).Osteoarthritis and Cartilage 2013;21(1):16-21.
2. Sharma A, Jagga S, Lee S, Nam J. Interplay between cartilage and subchondral bone contributing to pathogenesis of osteoarthritis.International journal of molecular sciences 2013;14(10):19805-19830.
3. Neogi T, Zhang Y. Epidemiology of Osteoarthritis. Rheumatic Disease Clinics of North America. 2013;39(1):1-19.
4. Losina E, Walensky R, Reichmann W et al. Impact of obesity and knee osteoarthritis on morbidity and mortality in older Americans.Annals of internal medicine 2011;154(4):217-226.
5. Meretoja V, Dahlin R, Kasper F, Mikos A. Enhanced chondrogenesis in co-cultures with articular chondrocytes and mesenchymal stem cells.Biomaterials 2012;33(27):6362-6369.
6. Ylöstalo J, Bartosh T, Coble K, Prockop D. Human mesenchymal stem/stromal cells cultured as spheroids are self‐activated to produce prostaglandin E2 that directs stimulated macrophages into an anti‐inflammatory phenotype.Stem cells 2012;30(10):2283-2296.

Adam Olson

Introduction. Damage to articular cartilage often results from physical trauma or age-related abrasion.⁴ In all cases, however, it leads to constant pain and functional limitations and can predispose individuals to osteoarthritis.^1,2,4 Once damaged, the poorly vascularized tissue is slow to repair and often heals improperly.^1-4

Many of the current treatments are centered on slowing disease progression and managing pain, but studies show that mesenchymal stem cells have the potential to promote the actual regeneration of damaged articular cartilage.

By analyzing the external signals that promote progenitor stem cells to differentiate down specific cell lineages, researchers are have successfully caused adipose-derived mesenchymal stem cells (AD-MSCs) to differentiate into articular cartilage ex vivo.^2,4,7 This study focuses on comparing intra-articular injections of AD-MSCs with micro-scaffolding and hyaluronan to promote stem cell differentiation in vivo.^3,6,8,10

Methods. Intra-articular Injections: 18 patients with osteoarthritis were randomized into 3 dose-escalated cohorts for arthroscopy and AD-MSCs injections into the knee. Patients were evaluated clinically at one month intervals following injection. Second look arthroscopy and MRI were performed at 6 months.³

Adherence and Proliferation: Ultra low adherence 96-well plates were plugged with cartilage discs and MSCs were seeded, suspended in either control or hyaluronan media (0.5–5mg/mL HA). Cells in each media were counted after 24 hours for adherence and after three days for proliferation.¹⁰ In a separate test, bone marrow and adipose-derived MSCs were seeded onto Chondro-Gide and Alpha Chondro Shield micro-scaffolding. Viable stem cells were counted after 24 hours, and again after one, two, and four week intervals.⁸

Results. Intra-articular injection of AD-MSCs did not lead to differentiation or proliferation of injected cells on its own.³ Instead, injected MSCs upregulated endogenous cells leading to an increase in healing of damaged tissues, increase in overall function of the joint, and decrease in pain on movement for patients receiving treatment.^3,6

In contrast, the use of Chondro-gide micro-scaffolding and the priming of AD-MSCs with hyaluronan both led to greater adherence of implanted stem cells to ex vivo cartilage as well as increased proliferation after adherence.^8,10 

Conclusion. The ability to influence AD-MSCs to differentiate down specific pathways towards chondrocyte formation was an important step in the successful regeneration of articular cartilage in vitro.^2,4,7,8,10 Micro-scaffolding and hyaluronan priming show additional potential in the transition from ex vivo to in vivo regeneration of articular cartilage. With additional study, AD-MSCs may finally lead to effective medical interventions for joint damage and osteoarthritis.

1. Akpancar, Serkan, et al. “The Current Perspectives of Stem Cell Therapy in Orthopedic Surgery.” Archives of Trauma Research 5.4 (2016): n. pag. Web. 15 Mar. 2017.
2. Guzzo, Rosa M., and Michael B. O’Sullivan. “Human Pluripotent Stem Cells: Advances in Chondrogenic Differentiation and Articular Cartilage Regeneration.” Current Molecular Biology Reports 2.3 (2016): 113-22. Web. 22 Mar. 2017.
3. Jo, Chris Hyunchul, et al. “Intra-Articular Injection of Mesenchymal Stem Cells for the Treatment of Osteoarthritis of the Knee: A Proof-of-Concept Clinical Trial.” Stem Cells 32.5 (2014): 1254-266. Web. 22 Mar. 2017.
4. Wu, Ling, et al. “Regeneration of articular cartilage by adipose tissue derived mesenchymal stem cells: Perspectives from stem cell biology and molecular medicine.” Journal of Cellular Physiology 228.5 (2013): 938-44. Web. 22 Mar. 2017.
5. Bari CD, Dell’accio F, Luyten FP. Failure of in vitro-differentiated mesenchymal stem cells from the synovial membrane to form ectopic stable cartilage in vivo. Arthritis & Rheumatism. 2004;50(1):142-150.
6. Black LL, et al. Effect of Adipose-Derived Mesenchymal Stem and Regenerative Cells on Lameness in Dogs with Chronic Osteoarthritis of the Coxofemoral Joings: a Randomized, Double-Blinded, Multicenter, Controlled Trial. Veterinary Therapeutics. 2007;8(4):272-284.
7. Casado-Díaz A, Anter J, Müller S, Winter P, Quesada-Gómez JM, Dorado G. Transcriptomic Analyses of Adipocyte Differentiation From Human Mesenchymal Stromal-Cells (MSC). Journal of Cellular Physiology. 2016;232(4):771-784.
8. Kohli N, Wright KT, Sammons RL, Jeys L, Snow M, Johnson WEB. An In Vitro Comparison of the Incorporation, Growth, and Chondrogenic Potential of Human Bone Marrow versus Adipose Tissue Mesenchymal Stem Cells in Clinically Relevant Cell Scaffolds Used for Cartilage Repair. Cartilage. 2015;6(4):252-263.
9. Rey-Rico A, Reinecke J, Wehling P, Cucchiarini M, Madry H. Effects of exosomes upon the metabolic activities of human osteoarthritic articular cartilage in situ. Osteoarthritis and Cartilage. 2015;23.
10. Succar P, Medynskyj M, Breen EJ, Batterham T, Molloy MP, Herbert BR. Priming Adipose-Derived Mesenchymal Stem Cells with Hyaluronan Alters Growth Kinetics and Increases Attachment to Articular Cartilage. Stem Cells International. 2016;2016:1-13.