Cartilage Tissue Engineering

John HenenSucheta Goyal

Cartilage Tissue Engineering

What is cartilage?

Cartilage function.

Damaged Cartilage has limited capacity for self-repair.

Causes for cartilage damage: Trauma Degenerative disease e.g. Arthritis .

Introduction http://enwikipedia.org/wiki/Cartila.ge

Dr Brian A Rothbart, 2010

Arthritis is a joint inflammation disorder. Inflammation happens due to cartilage degradation.No cure All treatments are to decrease symptoms or delay progress. Cartilage degradation may happens due to collagen degradation.

o Collagen puts the ECM together.

Background www.medicinenet.com

17.1% of the disabilities in USA because of arthritis. [Healthy people]

350 million people worldwide have arthritis. 37 million people in USA.

wrongdiagnosis.com

Health care: $81 billion annually in USA. abcnews.go.com

Rational

Using chondrocytes cells for cartilage repair. Chondrocytes are cells that build and maintain cartilage.Isolating and seeding of chondrocytes cells found from porcine

knees of patient. The cells are mature differentiated chondrocytes or

osteochondral progenitor cells, such as MSCs. (Huckle, et al. 2003)

Tissue-engineering Solution and rational (Method 1)

http://saveyourself.ca/articles/shorts/2009-05-26-carticel-good-news-bad-news.php

Chondrocytes: the cells that build and maintain cartilage

Isolating and seeding of chondrocytes: The cartilage tissue was harvested from porcine knees.

Preparation of protein-loaded chitosan microsphere: Solution mixed with FITC-BSA (fluorescein isothiocyanate bovine serum albumin) or TGF-β1(transforming growth factor –beta1).

Preparation of chitosan scaffold containing MS-TGF

SEM (scanning electron microscopy) measurement:-Chondrocytes were growing on the scaffold.

( Sung Eun Kim, et al. 2006)

Methods and Techniques

Chitosan microsphere loaded with proteins, BSA (bovine serum albumin) and TGF (transforming growth factor)in the presence of TPP (Thiamine pyrophosphate)

The size of microspheres was reduced by the protein.

Diffusion of TPP into microspheres is time dependent and critical to maintain the shape of microsphere .

The cross-linking time should be longer than 20 min.

(Sung Eun Kim, et al. 2006)

Results

In vitro protein release from microspheres:

The proteins, BSA (bovine serum albumin) and TGF (transforming growth factor) were released in biphasic manner.

implies that initially the rate of release is fast and later the release is slower.

BSA and TGF released at initial phase, 48.8% and 14.5% respectively.

Release rate of TGF-beta 1 from microspheres was lower than BSA over the whole experiment

BSA (90.1%) released within 5 days.Release amount of TGF- beta1 only

44.9% for 7 days.

Release profiles of FITC–BSA (0) and TGF-β1 from chitosan microspheres .

Results cont’d

( Sung Eun Kim, et al. 2006)

SEM (scanning electron microscopy) used for evaluation cell morphology and proliferation.

After 4 days, chondrocytes were present in the superficial area of the scaffold and maintain a spherical morphology.

After 7 days, chondrocytes were slightly proliferated without spreading.

Chondrocytes growth on chitosan scaffolds without microsphere-TGF

Scanning electron micrographs of chondrocytes grown on chitosan scaffold for (a) 4 days and (b) 7 days after seeding.

Results cont’d

( Sung Eun Kim, et al. 2006)

After 4 days , chondrocytes cells grow on chitosan scaffold with MS-TGF, there was a significant increase in the number. After 7 days , chondrocytes were proliferated and open pores of scaffold occupied by the numbers of aggregates, composed of several chondrocytes.

Chondrocytes growth on chitosan scaffolds with microsphere-TGF

Scanning electron micrographs of chondrocytes grown on chitosan scaffold, seeded with chitosan microsphere containing TGF-β1. (a) Four days after seeding; the cells were merged to form large aggregates adherent to the scaffold. (b) Magnified view of a representative chondrocyte grown for 4 days. (c) Seven days after seeding

Results cont’d

( Sung Eun Kim, et al. 2006)

TGF-β1, has promote the protein synthesis and cell proliferation in articular cartilage. It acts as an autocrine regulator of chondrocytes.

It also inhibits the actions of matrix metalloproteinase that play an important role in the digestion of the ECM in both normal and degenerative articular cartilage. ( Sung Eun Kim, et al. 2006).

Milestones

Using MSCs stem cells for cartilage repair. Implant MSCs encapsulated collagen based

scaffold. Stem cells found in all multi cellular

organisms. They can renew themselves and differentiate to different cell types.

Two types of stem cells:Embryonic stem cellsAdult stem cells

Tissue-engineering Solution (Method 2)

Adult stem cells are undifferentiated cells that are found in bone morrow, blood stream, retina and cornea of the eyes. (Bigaria, et. al 2006)

Adult stem cells

http://www.stemcellresearch.org/testimony/prentice.htm

MSCs is found in the bone marrow.MSCs can differentiate to many different cell types: (Bobis, et. al 2006)

Chondrocytes ( Cartilage)Osteoblasts (Bone)Myocytes (Muscle)

MSCs don’t trigger the immune response. (Bonfield, et. al 2010)MSCs are immunosuppression. (Patel, et. al

2008)

Mesenchymal stem cells

Implanting MSCs in a rabbit cartilage defect.MSCs encapsulated in collagen hydrogel (CH) and collagen

alginate hydrogel (CAH) scaffolds.The scaffold encapsulated in five groups of champers:

CH + TGF-Beta1 CH - TGF-Beta1 CAH + TGF-Beta1 CAH - TGF-Beta1 MSCs without any scaffolds nor growth factors

The champers implanted in the rabbits for 8 weeks. After 8m weeks, the rabbits sacrificed and champers

collected.

Methods (Zheng, et. al 2010)

MSCs without any scaffolds, no tissue found.

HE staining of sectioned tissue inside diffusion chamber showed:

(A) CH –TGF-b1 group showed cells of

round shape and high cell density.

(B) CAH –TGF-b1 group showed lower

cell density of round shape.

(C) CH +TGF-b1 group showed cells of

round shape and high cell density.

(D) CAH +TGF-b1 group showed cells of

round shape and high cell density.

Results

Immunohistochemical examination of sectioned tissue inside

diffusion chamber showed:

(A, E) CH –TGF-b1 group showed intense positive staining of type II collagen.

(B, F): CAH –TGF-b1 group showed less positive staining of type II collagen.

(C, G) CH +TGF-b1 group showed highly positive staining of type II collagen.

(D, H) CAH +TGF-b1 group showed highly positive staining of type II collagen.

Results cont’d

MSCs can differentiate to chondrocytes without interact with body fluids.

A scaffold must be used for chondrogenesis to happen.

CH support more chondrogenesis than CAH.

TGF-b1 is important for MSCs differentiation.

Milestones

Tissue engineered chondrocytes can be used to treat cartilage damage.MSCs can be used to treat cartilage damage:

Reduce the cost of healthcare for arthritis patients. Give arthritis patients definitive treatment. Open the way to treat other diseases.

Limitation: TGF-β1 appears to be a powerful molecule to repair

damaged cartilage. But high dose of intra-articular injection may induce chemotaxis and activation of inflammatory cells, resulting in fibrosis and osteophyte formation in cartilage defects.

Number of surgeries that should be done to cover all the affected areas.

Future implications

Which better to use chondrocytes or MSCs?

Do you think using these methods will be effective to treat arthritis?

Discussion

Bagaria, V., et al., Stem cells in orthopedics: current concepts and possible future applications. Indian J Med Sci, 2006. 60(4): p. 162-9.

Bobis, S., D. Jarocha, and M. Majka, Mesenchymal stem cells: characteristics and clinical applications. Folia Histochem Cytobiol, 2006. 44(4): p. 215-30.

Bonfield, T.L., et al., Defining human mesenchymal stem cell efficacy in vivo. J Inflamm (Lond), 2010. 7: p. 51.

Patel, S.A., et al., Immunological properties of mesenchymal stem cells and clinical implications. Arch Immunol Ther Exp (Warsz), 2008. 56(1): p. 1-8.

Zheng, L., et al., Chondrogenic differentiation of mesenchymal stem cells induced by collagen-based hydrogel: an in vivo study. J Biomed Mater Res A, 2010. 93(2): p. 783-92.

sung eun kim , et al.Porous chitson scaffold containing microspheres loaded with transforming growth factors-beta1: implications for cartilage tissue engineering: Biomedical Research Center, 19 May 2003.

Huckle, et al. Differentiated chondrocytes for cartilage tissue engineering: 2003.

References