A novel therapeutic modality using ... - diponed.com · DiponEd - TPC cooperation Dr. Bhaskar Vyas,...
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A novel therapeutic modality using
autologous Adipose derived Stromal
Vascular Fraction (SVF) for treating
Osteoarthritis knee
Presented by-
DiponEd - TPC cooperation
Dr. Bhaskar Vyas, Plastic Surgeon, Stem Cell Scientist
Dr. Rajni Vyas, Gynaecologist, Stem Cell Scientist
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Introduction
• Collaborators
1. M.S. University, Baroda
2. Centre for Cellular And Molecular Platforms
3. Manipal Institute of Regenerative Medicine
4. Samanvaya Trust
• Team
• The Presenters
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Collaborators
DiponEd TPC Varoda
From L to R: Prof. Ramchandran, Prof. Bharat Chattoo, Dr.
Ramesh Bhonde, Dr. Bhaskar Vyas, Dr Rajni Vyas and, Dr.
Anant Marathe
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Participants in the project
• Prof. Dr. Pradeep Mehta, Orthopedic Surgeon
• Dr. Atul Shah, Plastic Surgeon
• Dr. Umang Gandhi, Orthopedic Surgeon
• Dr. Anant Marathe, Technical Director
• Dr. Palak Patel, Radiologist, SSG Hospital
• Ms. Dhwani Trivedi, Physiotherapist
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Genesis of a novel protocol
• MSCs derived from Bone-marrow was gold standard till recently.
• ADMSCs are emerging as a platinum standard since current decade and are vastly investigated for regeneration.
• SVF derived from autologous AT is just emerging as a preferred mode of therapy with abundant literature support. This is in consonance with ICMR guidelines 2013.
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Genesis of a novel protocol
1. We obtained MSCs from bone-
marrow. Found that the % was ~ 1-
10 %.
2. We translated MSCs to cartilage.
This would mean therapeutically
they will lose stemness.
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Genesis of a novel protocol
• We were funded by DBT to isolate and culture MSCs obtained from 22 samples of AT. This was successfully done and, was awarded best research award at BIRAC III.
• We presented the data at Sir Ganga Ram Hospital with Dr. Prof. N. K Ganguly in Chair in 2015. In brief, the following images verify that AT yielded multipotent MSCs having properties for translineage translation too.
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Cartilage translated from ADMSCs
Genesis of a novel protocol
Chondrocytes
stained with
Alcian Blue stain ADMSCs
ADMSCs
translated to
chondrocytes
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Adipose Derived Mesenchymal Stem
Cells (ADMSCs)
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ADMSCs Positive Marker Studies
CD - 44 CD - 90
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ADMSCs Positive Marker Studies
CD - 29 CD - 105
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ADMSCs Negative Marker Studies
CD - 45 CD - 34 HLA-DR - ve
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Trans-differentiation of MSCs
Mesoderm - Chondrogenesis Ectoderm – Neurological
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Trans-differentiation of ADMSCs
C-Peptide Insulin Secreting Cells
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Genesis of a novel protocol
• We were awarded Biotechnology Ignition Grant (BIG) from BIRAC, DBT, GoI to establish a novel protocol for treatment of Osteoarthritis with Stromal Vascular Fraction (SVF).
• Between 2013 till today the focus has shifted from application of ADMSCs to AT-SVF with large literature support. This includes therapy for Osteoarthritis too.
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Genesis of a novel protocol
• The clinical trial began in June 2015. IEC & IC-
SCR approvals were obtained. DCGI approval
was obtained.
• The trial concluded in Nov 2016. The study is
submitted for publication to the Journal
Cytotherapy. A copy of the manuscript is
circulated.
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Animal Studies – not needed
• Abundant literature support in several species of animals from murine to caprine. Ref;
• Journal of Medical Engineering Volume 2013 (2013), Article ID 832396, 7 pages http://dx.doi.org/10.1155/2013/832396 Research Article
Transplantation of Nonexpanded Adipose Stromal Vascular Fraction and Platelet-Rich Plasma for Articular Cartilage Injury Treatment in Mice Model
Phuc Van Pham, Khanh Hong-Thien Bui, Dat Quoc Ngo, Lam Tan Khuat, andNgoc Kim Phan1Not needed as per latest GoI guidelines for research.
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Evolution to the present study; in
process
• With the above academics a study was
designed. A pilot project with six patients was
successfully carried out between 2012 – 2013.
This was presented to the expert committee
at BIRAC, DBT, GoI. The study is published in
Stem Cell: Advanced Research & Therapy.
Hard copy is circulated.
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Properties of MSCs in AT
1. Anti- apoptosis
2. Angiogenesis
3. Secretion of growth factors
4. Anti-fibrosis
5. Anti- inflammatory
6. Immunonaive
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Composition of SVF
1. MSCs – 30-40 %
2. CD 34+ haemopoietic stem cells
3. Endothelial precursor cells
4. T-regulatory cells
5. Macrophages
6. Smooth muscle cells
7. Pre-adipocytes
It is established that cells in SVF support regeneration of the cartilage.
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Latest references for SVF
• Andrew Ngyuen, James Guo. et al, Stromal Vascular fraction: A regenerative reality? Part 1: Current concepts and review of the literature, Journal of Plastic Reconstructive & Aesthetic Surgery 2015, xx,1e10.
• Philippe Bourin, Bruce A. Bunnell et. al, Stromal Cells from the adipose tissue-derived stromal vascular fraction and culture expanded adipose tissue-derived stromal/stem cells: a joint statement of the International Federation for Adipose Therapeutics (IFATS) and Science and the International Society for Cellular Therapy (ISCT), Cytotherapy 2013, 15 (6): 641-48.
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Flow Cytometric Analysis of SVF at Manipal
Institute of Regenerative Medicine
Fluorescence Tag Markers % Gated (M2) Result
PE Isotype Control 0.39
CD 90 39.81 Positive
CD 34 12.58 Positive (very few
cells)
FITC HLA DR 38.45 Positive
Isotype Control 1.37
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SVF Flowcytometry with CD90 at MIRM
MSCs % as 31+ % B
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SVF Flowcytometry with CD 34 at MIRM MSCs % as 12+ % B
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Denial of Disinformation
1. Stem cell are tumorogenic.
• Denied since autologous minimally manipulated ADMSCs and AT-SVF have no such incidence.
2. Multipotent stem cells may undergo translineage translation
• Denied since autologous minimally manipulated ADMSCs and AT-SVF have no such incidence.
There is only a single incidence of bone formation at the transplanted bone-marrow stem cells.
3. Large literature support for stability of genes.
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Cytokines in Platelet Rich Plasma
• Abundant literature support; given as handout
• platelet-derived growth factor
• transforming growth factor beta
• fibroblast growth factor
• insulin-like growth factor 1
• insulin-like growth factor 2
• vascular endothelial growth factor
• epidermal growth factor
• Interleukin 8
• keratinocyte growth factor
• connective tissue growth factor
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Study Design : Single centre Phase II randomised trial.
Advertisement
Response 138 patients
Pathology
investigations &
Cardiac reports
Suitable for
study as per
inclusion
criteria Non Suitable
as per
exclusion
criteria
Clinical examination
as out patients by
Orthopedic Surgeon
Written as well as audio
visual Consent Signing
Admission at the
hospital on the prior
evening Next day morning
Lipoaspiration by plastic
surgeon
Transfer of lipoaspirate to laboratory
for processing ~ 6 hrs. Processed
SVF transferred to OT
Injection of SVF and PRP by
Orthopedic Surgeon.
Pt discharged on the next
day. Bed rest for 15 days.
Gradual mobilization to
walk up to 1 km after 1
month.
Follow-up with
WOMAC & KSS for 1,
6 and 12 months
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Inclusion Criteria
• Able to express free will and give an informed
consent and willingness for 12 months follow
up.
• Proof of cartilage damage in the joint(s)
• Age between 50 to 80;
• Metabolic disorders such as hypothyroidism,
diabetes or abnormal blood pressure level to
be controlled before the initialization of
treatment.
• Patients showing Kellgren-Lawrence Grading
Scale of 2, 3 & 4.
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• Patients with loose bodies in the joint
• History of taking corticosteroids or NSAIDs, glycosaminoglycan
• Patients positive for Hepatitis B, C, HIV, viral or fungal infection
• History of allergic reactions
• History of severe concurrent medical condition eg: heart disease, lung disease, or hematopoietic dysfunction or liver dysfunction, or kidney
dysfunction.
Exclusion Criteria
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Included after correcting Associated Disease
Associated Diseases No. of Patients
High Blood Pressure 7
Hypothyroid 3
Diabetes 6
Previous Angioplasty 1
Previous Angiography 1
Medical condition was treated with drugs. Treatment
was done after the control of the disease.
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Pathological Investigations
Pathological Investigations
Hb HBsAg
CBC HCvAb
Creatinin HBA1c
SGPT FBS- PPBS
Prothrombin
Time (INR)
Urine Routine & Microscopy
Blood Group HIV
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BMI of 25 Patients
less than 20 Under wt. 1pt
21-25 Normal 8pts
26-30 Over wt. 10pts
31-35 obese 5pts
40 and more Very obese 1pt
Sex of Patients
female 18
male 7
Profile of patients R
Grade No. of knees
Grade 1 0
Grade 2 10
Grade 3 36
Grade 4 4
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MRI findings No. of Patients
MMT= Medial Meniscus tear 22
LMT= Lateral Meniscus tear 8
EMJ= Extrusion of meniscus joints 0
ACL= Anterior cruciate ligament 19
PCL= Posterior cruciate ligament 6
MCL= Medial Collateral ligament 0
LCL= Lateral Collateral ligament 0
CYST 14
OSTEO= Osteophytes 20
ISB= Infusion in suprapatellar bursa 20
SFTJ= Subluxation in femur-tibial joint 6
FB= Foreign bodies 4
MRI findings B
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WOMAC Score Statistical Analysis with ANOVA (Analysis of
Variance) one way and Tukey test (comparison between 2
parameters) showing significant improvement:
p value <0.001 (highly significant)
* p<0.05, **p<0.01 , ***p<0.001 Pre vs Post 1,6 and 12 months
#p<0.05, # #p<0.01, # # # p<0.0011 month vs 6 and 12 months
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KS Score Statistical Analysis with ANOVA one way and tukey test showing
significant improvement.
Inference – Significant improvement after 12 months
P value <0.05
* p<0.05, **p<0.01 , ***p<0.001 Pre vs Post 1,6 and 12 months
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* p<0.05, **p<0.01 , ***p<0.001 Pre vs Post 1,6 and 12 months
#p<0.05, # #p<0.01, # # # p<0.0011 month vs 6 and 12 month
Highly significant improvement. Inference – p value <0.001 R
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* p<0.05, **p<0.01 , ***p<0.001 Pre vs Post 1,6 and 12 months
#p<0.05, # #p<0.01, # # # p<0.0011 month vs 6 and 12 month
Highly significant improvement. Inference – p value <0.001 R
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* p<0.05, **p<0.01 , ***p<0.001 Pre vs Post 1,6 and 12 months
Highly significant improvement. Inference – p value <0.001
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Advantages at SGRH
• The project will be known as SGRH – Vadodara protocol.
• A leap forward in the most researched area in stem cell therapy in present decade.
• Will help serve poor even better than as of now. Subsidy will be low and project will attract donations.
• Certain to attract medical tourism.
• For the Orthopedic surgeons:
• Huge saving of operating time
• Additional tool in the armamentarium to help even health compromised
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Why the project needs SGRH
• The project has national relevance.
• GoI funds need to be deployed to yield dividends for societal benefit.
• At affordable price to most patients.
• It will be only second to Stempeutics stem cell product from India.
• It will be an in house product at SGRH – a la carte – autologous, easy to obtain.
• In full compliance with ICMR guidelines 2013.
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Futuristic unfolding at SGRH
• Other Orthopedic conditions – AVN femoral
head and Osteoarthritis elsewhere: cartilage
injuries and ligamentous tears.
• Endodermal translation for Diabetes I & II.
• Neuronal translation for neuro – restoration in
neurodegenerative diseases including
Parkinson’s disease.
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Conclusion
• Autologous SVF and PRP is a new potential
strategy to treat OA of human knees.
• With intra-articular injection of autologous
adipose derived SVF and autologous PRP, all
patients except one drop out improved.
• Further studies are necessary. Intra-articular
injections of autologous adipose SVF and
autologous PRP present a promising minimally
invasive option of treating OA successfully
with certain safety and high efficiency.
B
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Team Members
From L to R: Shivani Vakodikar, Dhwani Modi,
Jaymesh Thadani, Sandeep David, Prashant
Kshatriya and Dr. Nirupa Vyas
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