An Evaluation Of Amniotic Membrane Allograft As A Potential Agent For Biological Augmentation

Of Rotator Cuff Repair

Adnan Saithna1, Jennifer Z. Paxton2, Uchena Wudebwe2, Liam M. Grover2 and Martyn Snow1

1The Royal Orthopaedic Hospital2School of Chemical Engineering, The University of Birmingham

AMA and tendon repair

• Reduction of peri-tendinous adhesions (Ozgenel at al 2004 & Demirkan et al 2002)

• Favourable influence on collagen deposition (Yang et al 2010)

• Improved mechanical properties (Yang et al 2010, Ozbuluk et al 2010, He at al 2002, Ozgenel et al 2001)

Micronised AMA

Cellular Proliferation Study

0% SerumLow A.M. High A.M.

Images taken on Day 7

Manufacture and Maintenance of “Bone-Tendon” Constructs

Manufacture:- 35mm Sylgard coated Petri dish- Brushite anchor (Bone substitute)- Fibrin Gel (soft tissue substitute)- Seeded with 100,000 Chick Tendon Fibroblastss- DMEM + 10% FBS, 2.4% L-glutamine, 2.4% HEPES- 1% penicillin/streptomycin

Maintenance:- Incubated at 37°C, 5% CO2

- Fed with s-DMEM every 2-3 days

Ascorbic acid (250µM) and proline (50µM) were added on day 7 onwards, along with AMA (13µl/ml DMEM) in the treatment group.

Typical sequence of maturation

AMA Dosing

• Cost prohibitive

• “One vial per cuff” strategy

• Calculation based on previous work on surface area of soft tissue attachment to the brushiteanchor (Paxton et al, Ann Biomed Eng, 2010)

• Rotator cuff footprint area (Mochizuki et al, JBJSAm 2009)

• 13uL per ml of DMEM

Fibrin Gel Contraction• Constructs photographed daily and image J analysis

software was used to assess area and rate of contraction. No significant difference was demonstrated between control and treatment groups

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Area of constructmm2

Day of Culture

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Mechanical Testing

• Instron Microtester

– 37oC Water Bath

– 10N Load Cell

– Load applied at 0.4mm/sec

– Load/Extension and mode of failure recorded

Mechanical Testing - Results

• The mean maximum load to failure of the hard/soft interface in the control group was 0.33N (+/-0.24) and 0.23N (+/-0.10) in the AMA group. No statistically significant difference was demonstrated between groups (p=0.371).

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Control AMA

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Treatment

Collagen content

• There was no difference between groups with respect to ug of hydroxyproline per mg of tissue Control 35.76ug +-10.19, AMA 31.7ug +/- 8.49, p=0.72

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Discussion

• AMA had no significant effect on cellular proliferation , collagen content, or on the mechanical properties of the interface.

• In contrast to previous work. However these animal studies used human amniotic fluid or membrane rather than a micronized preparation and also investigated mid-substance tendon healing rather than the specifically investigating the bone/tendon interface.

Limitations

• Dose of AMA restricted by cost

• In vitro study – so not the whole picture

• Model only evaluates fibroblasts

• No inflammatory component

Future Directions

Localised AMA delivery

- Anchor encased in 150uL of fibrin containing a single dose of 26uL of AMA

Mechanical Testing - Localised AMA

• A trend towards increased maximum load to failure was seen in the AMA group but this was not significant (control 0.20N (+/- 0.13), fibrin 0.21N (+/-0.02), fibrin + AMA 0.29N (+/-0.02)

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Control Fibrin Fibrin+AMA

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Treatment

Conclusions

• No benefit of AMA at a maximum cost effective dose for clinical application.

• Model allows easy in vitro assessment of potentially therapeutic agents but has some limitations.