Re: [Histonet] maximize the hardness of MMA

From:"Dr. Adam Hacking, PhD"

Dear all,

Thank you for your responses. Pam as requested here is some background information. Please don't fall asleep :-)

I have a technique where I coat a nylon rod with a homogeneous sub micron thick layer of titanium. These rods are implanted in the femurs of rats or mice and the tissue response to the implant is assessed. The goal of the model is to assess tissue and cell response to titanium implants using traditional light microscopy techniques (usually we just asses bone via backscattered electron microscopy [BSEM]) 

The model works quite well, and as far as the tissue response is concerned the tissue responds as if it were a solid titanium implant. One ongoing problem is separation of the methylmethacrylate (and tissue) from interface with the nylon rod during sectioning.   

I am very familiar with embedding Ti implants and sectioning with a diamond saw  however microtome sectioning is new to me (thankfully we do have a very capable technician who is familiar with MMA embedding and sectioning) but offers a number of tests that cannot be performed by BSEM.  

I tried a batch of MMA that was activated by addition of benzoyl peroxide [BPO] and this seemed harder than our tech's low temp method of embedding. The sections were much better and I attributed this to the additional hardness of the resin and its resistance to compression during sectioning. However some of our tests (ALP, TRAP) did not work. We have tried increasing the BPO concentration and this has some effect. 

Karen, I will recommend skipping the n-butyl phthalate. 

Here are our mixtures of MMA. All are fixed in para formaldehyde for  48 hours. 

Harder MMA tested (classic implant MMA)

Dehydrate samples : 
75, 85, 95 % EtOH 48 hrs each

Defat 1:1 Ether : Acetone 48 hrs

Final Dehydrate 100% EtOH 48 hrs

Cold MMA (MMA with0.5% BPO) 48 hrs

Hot MMA (MMA with0.5% BPO and heated at 56 C for 4-5 hours) 
Embed under alternating vacuum 48 hrs

Low TEMP MMA (RG Erben The Journal of Histochemistry & Cytochemistry 1997)

75, 85, 95 % EtOH 48 hrs each
100% EtOH 24 hrs
100% Zylene 48 hrs

48 hrs - MMA Solution I consisted of 60 ml MMA (Merck; containing 100 ppmhydrochinon) 35ml butylmethacrylate (Sigma; containing 10 ppm hydrochinon) 5 ml methylbenzoate 1.2 ml polyethylene glycol 400. @ 4C

48 hrs - 100 ml MMA solution above plus with 0.4 g dry benzoyl peroxide@ 4C

Embedded - 100 ml MMA solution above plus with 0.8 g dry benzoyl peroxide @ 4C

Thank you again.

Adam Hacking

Pamela Marcum  wrote: Hi Adam,

The best way to have the hardest MMA is to use straight MMA, with no 
dibutyl phthalate.  It is a mixture of MMA as the plastic and a small 
amount of benzyol peroxide.  IF you can tell me (us) what you wish to 
embed and how you will section and stain it (as well as view light 
microscopy or EM) the information would make it easier to help you 
with the problem.

Pam Marcum

At 01:14 PM 1/2/2007, you wrote:
>Does anyone know how to increase or maximize the hardness of MMA?
>Are there harder embedding materials ?
>Many thanks
>Adam Hacking. PhD
>Dr. S Adam Hacking, Post Doctoral Fellow
>JTN Wong Laboratories for Mineralized Tissue Research,
>Center for Bone and Periodontal Research,
>McGill University     740 Dr. Penfield Ave. Rm. 2201
>Montreal, QC, Canada H3A 1A4
>Ph.:  514-398-5112
>Fax: 514-398-4020
>Histonet mailing list

Best Regards,

Pamela A Marcum
Manager, Histology Special Procedures
University of Pennsylvania
School of Veterinary Medicine
R.S. Reynolds Jr.  CORL
New Bolton Center
382 West Street Road
Kennett Square, PA 19348

Phone - 610-925-6278
Fax     - 610-925-8120
E-mail - 

________________________________________________________________         Dr. S Adam Hacking, Post Doctoral Fellow
JTN Wong Laboratories for Mineralized Tissue Research, 
Center for Bone and Periodontal Research, 
McGill University     740 Dr. Penfield Ave. Rm. 2201 
Montreal, QC, Canada H3A 1A4 
Ph.:  514-398-5112
Fax: 514-398-4020
Histonet mailing list

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