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Thank you to all who replied to my question on the effects of freezing
bone specimens. The original question is below with the summary of responses
following:
I would like to know if anyone has any information on the effects of
freezing on the mechanical properties of bone. I vaguely recall this
topic coming up on biomch-l some time ago but can't remember the details.
I am interested to find out whether single or multiple freeze/thaw cycles
affect the mechanical properties of small vertebrae for compression testing.
Do bones suffer "freezer burn" with subsequent dehydration, even if
wrapped in polythene cling film? Would it be better to freeze bones in
small wells of saline?
Talking of dehydration: If allowed to dehydrate, would the mechanical
properties of such a bone be restored to normal when rehydrated by submerging
it in physiological saline?
Responses:
1. You might write Dr. Martin Krag at the University of Vermont.
2. I recommend contacting Subrata Saha, Ph.D, Loma Linda University in
California. He should have some information on this important question.
3. Freezing, thawing and refreezing appear to have little effect upon the
mechanical properties of bone in general, and especially whole bones.
The earliest paper I know on the subject is: Sedlin, E.D. and Hirsch, C.,
1996; Factors affecting the determination of the physical properties of
femoral cortical bone. Acta orthop Scand 37:29-48.
Last year we published a paper on strain measurments in human mandibles
that had repeated freezing and thawing, and some drying and rehydrating,
and found no apparent changes in the mechanical properties: Throckmorton,
G.S. and Dechow, P.C., 1994: In vitro strain measurements in the condylar
process of the human mandible. Archs. oral Biol. 39:853-867.
Since then Dr. Paul Dechow and his students at Baylor College of
Dentistry has done some more studies along these lines. He can be reached
at pcdechow@metronet.com.
4. Freezing seems to be the best way to keep the mechanical properties of bone
constant. Sequences of thawing and freezing increase the risk of trabecular
damage by temperature expansion and freezing expansion of the water the bone
specimens contains.
The following literature could be of potential interest:
Sonstegard DA & Matthews LS Mechanical propertiy dependence on storage
technique and local of knee joint trabeculae Trans ORS 2:283, 1977
Pelker RR et al. Effects of freezing and freeze-drying on the biomechanical
properties of rat bone J Ortop Res 1405-411, 1984
Panjabi MM et al. Biomechanical time tolerance of fresh cadaveric human spine
specimens J Ortop Res 3: 292-300, 1985
5. Linde F, Sorensen HCF: The effect of different
storage methods on the mechanical properties
of trabecular bone. J Biomechanics 26:1249-1252, 1993
Pelker RR, Friedlaender GE, Markham TC, Panjabi MM, Moen CJ:
Effects of freezing and freeze-drying on the biomechanical
properties of rat bone. J Orthop Res 1:405-411, 1984
Each of these articles contains a few other references.
6. In addition to the references I supplied, I would
like to offer a few comments from experience regarding your
specific questions:
I do *not* think it is sufficient to wrap specimens in
cling film because a truly airtight seal is difficult
to achieve. With small specimens, I have taken a three-
fold approach: wrap specimens individually in cling
film and place in individual sealed vials, then store
groups of vials in zip-lock plastic bags.
I strongly caution you *against* freezing specimens
in small wells of saline because of (potentially
damaging) pressure which would be applied to
the bone when the liquid expands upon freezing.
I doubt that the mechanical properties of a "dehydrated"
bone as you described it would be restored by
merely soaking it in saline. This is because the
dehydration may cause contraction and subsequent
microdamage to the bone, which would be irreversible.
But I don't have any data to back up this idea.
7. I just finished my master's thesis in which I looked at the compressive
properties of rat vertebral bodies that had been dehydrated (not by me!) so
I looked into the effects of freezing and dehydration.
Pelker et al. [1984; see references below] reported that neither freezing
at -70 C nor freeze-drying affected the strength of rat vertebrae in
compression. They rehydrated the vertebrae in physiological saline for 24
hours at 4 C.
One set of the rat vertebrae that I used (from 16-30 week old female rats)
was kept frozen at -70 between (non-mechanical) tests, so they were thawed
and refrozen twice (rehydrated, frozen, thawed, tested, frozen, thawed, and
subjected to mechanical testing). I revised my protocol for the next set
(which were rehydrated and kept in the fridge until testing). However, I
did plot up my data for the repeatedly frozen set against data from other
researchers who had used fresh or frozen vertebrae, and found that the
strengths and stiffness were on the low side, but they were not out of
range of results from other groups, and they had comparable or smaller
standard deviations. Also, a significant part of the difference may be
accounted for by differences in mounting and testing protocols, which is a
major issue in the testing of these vertebrae.
I froze my bones wrapped in saline-moistened gauze in individual, air-tight
containers. I can't really tell you whether they suffered freezer burn or
not.
The literature reports that the torsion and bending properties of long
bones are reduced by freezing, freeze-drying or dehydration than the
compressive properties of vertebrae although my multiply-frozen long bones
had, again, strengths and stiffnesses that were midrange compared to
literature values for frozen or fresh bones.
Here are some references:
Freezing, freeze-drying and dehydration of bone:
Pelker RR, Friedlander GE, Markham TC, Panjabi MM, Moen CJ (1984) Effects
of freezing and freeze-drying on the biomechanical properties of rat bone.
J Orthop Res 1(4):405-411.
Currey JD (1988) The effects of drying and re-wetting on some mechanical
properties of cortical bone. J Biomechanics 21(5):439-441.
Stromberg L, Dalen N (1976) The influence of freezing on the maximum torqu=
e
capacity of long bones. Acta Orthop Scand 47:254-256.
And here are some studies looking at fresh/frozen vertebrae:
(these are the studies that I compared my data to)
Turner CH, Sato M, Bryant HU (1994) Raloxifene preserves bone strength and
bone mass in ovariectomized rats. Endocrinol 135:2001-2005.
Mosekilde L, Danielsen CC, Knudsen UB (1993) The effect of aging and
ovariectomy on the vertebral bone mass and biomechanical properties of
mature rats. Bone 14:1-6.
Mosekilde L, S=F8gaard CH, McOsker JE, Wronski TJ (1994) PTH has a more
pronounced effect on vertebral bone mass and biomechanical competence than
antiresorptive agents (estrogen and bisphosphonate)-assessed in sexually
mature, ovariectomized rats. Bone 15(4):401-408.
Lauritzen DB, Balena R, Shea M, Seedor JG, Markatos A, Le HM, Toolan BC,
Myers ER, Rodan GA, Hayes WC (1993). Effects of combined prostaglandin and
alendronate treatment on the histomorphometry and biomechanical properties
of bone in ovariectomized rats. J Bone Min Res 8(7):871-879.
Richard Hillam
bone specimens. The original question is below with the summary of responses
following:
I would like to know if anyone has any information on the effects of
freezing on the mechanical properties of bone. I vaguely recall this
topic coming up on biomch-l some time ago but can't remember the details.
I am interested to find out whether single or multiple freeze/thaw cycles
affect the mechanical properties of small vertebrae for compression testing.
Do bones suffer "freezer burn" with subsequent dehydration, even if
wrapped in polythene cling film? Would it be better to freeze bones in
small wells of saline?
Talking of dehydration: If allowed to dehydrate, would the mechanical
properties of such a bone be restored to normal when rehydrated by submerging
it in physiological saline?
Responses:
1. You might write Dr. Martin Krag at the University of Vermont.
2. I recommend contacting Subrata Saha, Ph.D, Loma Linda University in
California. He should have some information on this important question.
3. Freezing, thawing and refreezing appear to have little effect upon the
mechanical properties of bone in general, and especially whole bones.
The earliest paper I know on the subject is: Sedlin, E.D. and Hirsch, C.,
1996; Factors affecting the determination of the physical properties of
femoral cortical bone. Acta orthop Scand 37:29-48.
Last year we published a paper on strain measurments in human mandibles
that had repeated freezing and thawing, and some drying and rehydrating,
and found no apparent changes in the mechanical properties: Throckmorton,
G.S. and Dechow, P.C., 1994: In vitro strain measurements in the condylar
process of the human mandible. Archs. oral Biol. 39:853-867.
Since then Dr. Paul Dechow and his students at Baylor College of
Dentistry has done some more studies along these lines. He can be reached
at pcdechow@metronet.com.
4. Freezing seems to be the best way to keep the mechanical properties of bone
constant. Sequences of thawing and freezing increase the risk of trabecular
damage by temperature expansion and freezing expansion of the water the bone
specimens contains.
The following literature could be of potential interest:
Sonstegard DA & Matthews LS Mechanical propertiy dependence on storage
technique and local of knee joint trabeculae Trans ORS 2:283, 1977
Pelker RR et al. Effects of freezing and freeze-drying on the biomechanical
properties of rat bone J Ortop Res 1405-411, 1984
Panjabi MM et al. Biomechanical time tolerance of fresh cadaveric human spine
specimens J Ortop Res 3: 292-300, 1985
5. Linde F, Sorensen HCF: The effect of different
storage methods on the mechanical properties
of trabecular bone. J Biomechanics 26:1249-1252, 1993
Pelker RR, Friedlaender GE, Markham TC, Panjabi MM, Moen CJ:
Effects of freezing and freeze-drying on the biomechanical
properties of rat bone. J Orthop Res 1:405-411, 1984
Each of these articles contains a few other references.
6. In addition to the references I supplied, I would
like to offer a few comments from experience regarding your
specific questions:
I do *not* think it is sufficient to wrap specimens in
cling film because a truly airtight seal is difficult
to achieve. With small specimens, I have taken a three-
fold approach: wrap specimens individually in cling
film and place in individual sealed vials, then store
groups of vials in zip-lock plastic bags.
I strongly caution you *against* freezing specimens
in small wells of saline because of (potentially
damaging) pressure which would be applied to
the bone when the liquid expands upon freezing.
I doubt that the mechanical properties of a "dehydrated"
bone as you described it would be restored by
merely soaking it in saline. This is because the
dehydration may cause contraction and subsequent
microdamage to the bone, which would be irreversible.
But I don't have any data to back up this idea.
7. I just finished my master's thesis in which I looked at the compressive
properties of rat vertebral bodies that had been dehydrated (not by me!) so
I looked into the effects of freezing and dehydration.
Pelker et al. [1984; see references below] reported that neither freezing
at -70 C nor freeze-drying affected the strength of rat vertebrae in
compression. They rehydrated the vertebrae in physiological saline for 24
hours at 4 C.
One set of the rat vertebrae that I used (from 16-30 week old female rats)
was kept frozen at -70 between (non-mechanical) tests, so they were thawed
and refrozen twice (rehydrated, frozen, thawed, tested, frozen, thawed, and
subjected to mechanical testing). I revised my protocol for the next set
(which were rehydrated and kept in the fridge until testing). However, I
did plot up my data for the repeatedly frozen set against data from other
researchers who had used fresh or frozen vertebrae, and found that the
strengths and stiffness were on the low side, but they were not out of
range of results from other groups, and they had comparable or smaller
standard deviations. Also, a significant part of the difference may be
accounted for by differences in mounting and testing protocols, which is a
major issue in the testing of these vertebrae.
I froze my bones wrapped in saline-moistened gauze in individual, air-tight
containers. I can't really tell you whether they suffered freezer burn or
not.
The literature reports that the torsion and bending properties of long
bones are reduced by freezing, freeze-drying or dehydration than the
compressive properties of vertebrae although my multiply-frozen long bones
had, again, strengths and stiffnesses that were midrange compared to
literature values for frozen or fresh bones.
Here are some references:
Freezing, freeze-drying and dehydration of bone:
Pelker RR, Friedlander GE, Markham TC, Panjabi MM, Moen CJ (1984) Effects
of freezing and freeze-drying on the biomechanical properties of rat bone.
J Orthop Res 1(4):405-411.
Currey JD (1988) The effects of drying and re-wetting on some mechanical
properties of cortical bone. J Biomechanics 21(5):439-441.
Stromberg L, Dalen N (1976) The influence of freezing on the maximum torqu=
e
capacity of long bones. Acta Orthop Scand 47:254-256.
And here are some studies looking at fresh/frozen vertebrae:
(these are the studies that I compared my data to)
Turner CH, Sato M, Bryant HU (1994) Raloxifene preserves bone strength and
bone mass in ovariectomized rats. Endocrinol 135:2001-2005.
Mosekilde L, Danielsen CC, Knudsen UB (1993) The effect of aging and
ovariectomy on the vertebral bone mass and biomechanical properties of
mature rats. Bone 14:1-6.
Mosekilde L, S=F8gaard CH, McOsker JE, Wronski TJ (1994) PTH has a more
pronounced effect on vertebral bone mass and biomechanical competence than
antiresorptive agents (estrogen and bisphosphonate)-assessed in sexually
mature, ovariectomized rats. Bone 15(4):401-408.
Lauritzen DB, Balena R, Shea M, Seedor JG, Markatos A, Le HM, Toolan BC,
Myers ER, Rodan GA, Hayes WC (1993). Effects of combined prostaglandin and
alendronate treatment on the histomorphometry and biomechanical properties
of bone in ovariectomized rats. J Bone Min Res 8(7):871-879.
Richard Hillam