Functional properties of palmaris longus muscles of rhesus monkeys transplanted as index finger flexors

This experiment with skeletal muscle autografts in monkeys was designed to retest previous findings that transplanted skeletal muscle can regenerate to a functional degree in primates without predenervation and to test a new hypothesis that increased functional demands on regenerated muscle grafts in monkeys may result in improved functional capacity of the grafts. Rhesus monkey index flexors were replaced with free palmaris longus muscle autografts with microneural anastomoses between the graft motor nerve and the severed profundus motor nerve. One monkey was taught selective index flexion before grafting and continued with this program after grafting to test the effect of training on the graft. Mature grafts were evaluated for in vivo contractile properties and by histology and histochemistry and were compared with a group of normal Rhesus palmaris longus muscles. The results reconfirm the capacity of nonpredenervated monkey skeletal muscle grafts to regenerate and to achieve some contractile ability and suggest that training of free muscle grafts may enhance recovery of their functional and structural properties.     

"Donor" muscle structure and function after end-to-side neurorrhaphy

End-to-end nerve coaptation is the preferred surgical technique for peripheral nerve reconstruction after injury or tumor extirpation. However, if the proximal nerve stump is not available for primary repair, then end-to-side neurorrhaphy may be a reasonable alternative. Numerous studies have demonstrated the effectiveness of this technique for muscle reinnervation. However, very little information is available regarding the potential adverse sequelae of end-to-side neurorrhaphy on the innervation and function of muscles innervated by the "donor" nerve. End-to-side neurorrhaphy is hypothesized to (1) acutely produce partial donor muscle denervation and (2) chronically produce no structural or functional deficits in muscles innervated by the donor nerve. Adult Lewis rats were allocated to one of two studies to determine the acute (2 weeks) and chronic (6 months) effects of end-to-side neurorrhaphy on donor muscle structure and function. In the acute study, animals underwent either sham exposure of the peroneal nerve (n = 13) or end-to-side neurorrhaphy between the end of the tibial nerve and the side of the peroneal nerve (n = 7). After a 2-week recovery period, isometric force (F(0) was measured, and specific force (sF(0) was calculated for the extensor digitorum longus muscle ("donor" muscle) for each animal. Immunohistochemical staining for neural cell adhesion molecule (NCAM) was performed to identify populations of denervated muscle fibers. In the chronic study, animals underwent either end-to-side neurorrhaphy between the end of the peroneal nerve and the side of the tibial nerve (n = 6) or sham exposure of the tibial nerve with performance of a peroneal nerve end-to-end nerve coaptation approximately 6), to match the period of anterior compartment muscle denervation in the end-to-side neurorrhaphy group. After a 6-month recovery period, contractile properties of the medial gastrocnemius muscle ("donor" muscle) were measured. Acutely, a fivefold increase in the percentage of denervated muscle fibers (1 +/0 0.7 percent to 5.4 +/-2.7 percent) was identified in the donor muscles of the animals with end-to-side neurorrhaphy (p < 0.001). However, no skeletal muscle force deficits were identified in these donor muscles. Chronically, the contractile properties of the medial gastrocnemius muscles were identical in the sham and end-to-side neurorrhaphy groups. These data support our two hypotheses that end-to-side neurorrhaphy causes acute donor muscle denervation, suggesting that there is physical disruption of axons at the time of nerve coaptation. However, end-to-side neurorrhaphy does not affect the long-term structure or function of muscles innervated by the donor nerve.     

Effect of foreign innervation on contractile and histochemical properties of the transplanted levator ani muscle of the rat.

Heterotopic transplantation of the levator ani (LA) muscle into the bed of the fast tibialis anterior (TA) or slow soleus (SOL) muscle respectively results in transformation of contractile and histochemical properties of the muscle dependent on the new "foreign" innervation. This transformation is observed after transplantation of minced muscle tissue and of free grafts. The result of transformation is more pronounced in the case of free LA-TA grafts which show progressive shortening of contractile response, whereas the LA-SOL shows slight shortening. The heterotopically transplanted free LA-SOL and the LA-TA grafts become relatively faster than the respective original muscle, suggesting operation of myogenic factors related to the fast LA muscle. Maximal tetanic tension output of the free heterotopic grafts 60 days after transplantation recovers to only about a quarter of the correspondong control muscles. Recovery of speed of contraction in the transplanted LA muscle is similar to that observed after selfreinnervation after crushing the pudendal nerve close to its entry into the muscle. In the heterotopically transplanted muscles the reversal of the originally uniform histochemical fibre pattern to a mixed fibre pattern in respect to ATPase and SDH activity is dependent on the type of innervation. After selfreinnervation of the LA muscle by the pudendal nerve a uniform fibre pattern is maintained with regeneration of the nerve.     

Formation of muscle spindles in regenerated avian muscle grafts

Summary Pigeon muscles lacking muscle spindles were grafted into sites which normally have a muscle containing spindles. The reciprocal transplantations were also made. After two to eight months, the graft of the donor muscle without spindles had regenerated into a muscle containing muscle spindles. The reciprocal grafts, muscles containing spindles transplanted to a site lacking spindle innervation, had neither muscle spindles nor remnants of the spindles. These experiments demonstrate that 1) the innervation is required for formation of the spindle; 2) the original spindles do not survive transplantation; and 3) parts of the original spindle are not required for spindle regeneration.This work was supported in part by NSF grants PCM 77-15960 and PCM 79-16540     

Transplantation and transposition of skeletal muscles into the faces of monkeys

Restoration of normal facial movement after long-term facial paralysis with muscle atrophy has not yet been achieved reliably by either free grafts, in which fibers degenerate and regenerate, or by grafts made with microneurovascular repair, in which most fibers survive. Our purpose was to compare the structural and functional properties of free muscle grafts and continuously perfused muscle flaps transplanted into the faces of monkeys. In adult monkeys, the facial muscles were replaced by either a free graft of a donor muscle from the lower limb or a denervated flap of ipsilateral temporalis muscle. Each graft or flap was reinnervated with the preserved buccal branch of the facial nerve. The control muscles, grafts, and flaps were examined 90 days later for gross appearance, contractile properties, and fiber areas. Compared with muscle flaps, free grafts showed greater adaptability to the new location and innervation and a closer approximation to the structural and functional properties of the original facial musculature.     

Parallel-fibered muscles transplanted with neurovascular repair into bipennate muscle sites in rabbits.

Experiments were performed on 20 New Zealand White male rabbits. Our hypotheses were that (1) latissimus dorsi (LTD) muscles transplanted into the site of a bipennate rectus femoris (RFM) muscle with neurovascular repair would retain their parallel-fibered structure and (2) the parallel-fibered structure of latissimus dorsi grafts would reduce their total fiber cross-sectional area and adversely affect force development relative to that of bipennate rectus femoris grafts and muscles. Compared with their respective donor muscles, 120 to 150 days after grafting, latissimus dorsi and rectus femoris grafts showed no change in the number of fibers and a decrease in the mean single-fiber cross-sectional area to approximately 70 percent. The latissimus dorsi grafts, which remained parallel-fibered, developed maximum forces 34 and 23 percent of the values for fully activated rectus femoris grafts and muscles, respectively. The deficit in the maximum force of the latissimus dorsi grafts resulted primarily from the smaller total-fiber cross-sectional area as a result of the parallel-fibered structure.     

Tissue engineering of peripheral nerves: A comparison of venous and acellular muscle grafts with cultured Schwann cells

Bioengineering is considered to be the laboratory-based alternative to human autografts and allografts. It ought to provide "custom-made organs" cultured from patient's material. Venous grafts and acellular muscle grafts support axonal regeneration only to a certain extent because of the lack of viable Schwann cells in the graft. We created a biologic nerve graft in the rat sciatic nerve model by implanting cultured Schwann cells into veins and acellular gracilis muscles, respectively. Autologous nerve grafts and veins and acellular muscle grafts without Schwann cells served as controls. After 6 and 12 weeks, regeneration was assessed clinically, histologically, and morphometrically. The polymerase chain reaction analvsis showed that the implanted Schwann cells remained within all the grafts. The best regeneration was seen in the control; after 12 weeks the number of axons was increased significantly compared with the other grafts. A good regeneration was noted in the muscle-Schwann cell group, whereas regeneration in both of the venous grafts and the muscle grafts without Schwann cells was impaired. The muscle-Schwann cell graft showed a systematic and organized regeneration including a proper orientation of regenerated fibers. The venous grafts with Schwann cells showed less fibrous tissue and disorganization than the veins without Schwann cells, but failed to show an excellent regeneration. This might be attributed to the lack of endoneural-tube-like components serving as scaffold for the sprouting axon. Although the conventional nerve graft remains the gold standard, the implantation of Schwann cells into an acellular muscle provides a biologic graft with basal lamina tubes as pathways for regenerating axons and the positive effects of Schwann cells producing neurotrophic and neurotropic factors, and thus, supporting axonal regeneration.     

Quantification and origin of spindles in orthotopic and heterotopic grafts of avian muscles

Summary Regeneration of muscle spindles was quantified in a series of orthotopically and heterotopically autografted muscles of pigeons. Significantly fewer spindles relative to numbers of extrafusal fibers were present in grafts than in normal muscles. These results are in marked contrast to observations of free-grafted muscles of rats. A majority of grafts of the metapatagialis, a muscle devoid of spindles, into the site of the anterior latissimus dorsi contained spindles. A few spindles were present in grafts of the extensor digitorum communis, which normally contains many spindles, into the site formerly occupied by the metapatagialis whereas muscle spindles were absent in orthotopic grafts of the metapatagialis muscle. These observations suggest that the spindle-like structures observed in the extensor digitorum communis muscles, which regenerated in the sites of the metapatagialis, were derived from spindles of the donor muscle. Thus muscle spindles in transplanted avian muscle can form by two distinct developmental processes.This investigation was supported in part by research grants 1RO1AM26992 from the Public Health Service and PCM 79-16540 from the National Science Foundation     

Review: Whole skeletal muscle transplantation: mechanisms responsible for functional deficits

One aspect of tissue engineering of skeletal muscle involves the transposition and transplantation of whole muscles to treat muscles damaged by injury or disease. The transposition of whole muscles has been used for many decades, but since 1970, the development of techniques for microneurovascular repair has allowed the transplantation of muscles invariably result in structural and functional deficits. The deficits are of the greatest magnitude during the first month, and then a gradual recovery results in the stabilization of structural and functional variables between 90 and 120 days. In stabilized vascularized grafts ranging from 1 to 3 g in rats to 90 g in dogs, the major deficits are approximately 25% decrease in muscle mass and in most grafts approximately 40% decrease in maximum force. The decrease in power is more complex because it depends on both the average shortening force and the velocity of shortening. As a consequence, the deficit in maximum power may be either greater or less than the deficit in maximum force. Tenotomy and repair are the major factors responsible for the deficits.Although the data are limited, skeletal muscle grafts appear to respond to training stimuli in a manner no different from that of control muscles. The training stimuli include traditional methods of endurance and strength training, as well as chronic electrical stimulation. Transposed and transplanted muscles develop sufficient force and power to function effectively to: maintain posture; move limbs; sustain the patency of sphincters; partially restore symmetry in the face; or serve as, or drive, assist devices in parallel or in series with the heart.     

Limb muscle regeneration in peripheral nerve autografts

In a previous study we demonstrated regenerative growth of extraocular muscle within transplanted peripheral nerve autografts. The present study addresses the feasibility of inducing regeneration of limb muscle within autologous peripheral nerve implants in the gluteus medius of beagles. In six anesthetized animals, a 2-cm segment of the left infraorbital sensory nerve was removed from the nose and implanted between the cut ends of several muscle fascicles in the left gluteus medius. After 4 weeks, the nerve grafts were removed and examined by light and electron microscopy. Muscle fibers were seen surrounded by the epineurium of the implanted nerve along its entire length, growing in parallel with the long axis of the nerve. The regenerating fibers were closely associated with the basal lamina of degenerating myelinated and unmyelinated axons. This study suggests that limb muscle, like extraocular muscle, is capable of organized regenerative growth within peripheral nerve autografts.     

Differences in the Developmental Fate of Cultured and Noncultured Myoblasts When Transplanted into Embryonic Limbs

Myoblasts from embryonic, fetal, and adult quail and chick muscles were transplanted into limb buds of chick embryos to determine if myoblasts can form muscle fibers in heterochronic limbs and to define the conditions that affect the ability of transplanted cells to populate newly developing limb musculature. Myoblasts from each developmental stage were either freshly isolated and transplanted or were cultured prior to transplantation into limb buds of 4- to 5-day (ED4-5) chick embryos. Transplanted myoblasts, regardless of the age of the donor from which they were derived, formed muscle fibers within embryonic limb muscles. Transplanted cloned myoblasts formed muscle fibers, although there was little evidence that the number of transplanted myoblasts significantly increased following transplantation or that they migrated any distance from the site of injection. The fibers that formed from transplanted clonal myoblasts often did not persist in the host limb muscles until ED10. Diminished fiber formation from myoblasts transplanted into host limbs was observed whether myoblasts were cloned or cultured at high density. However, when freshly isolated myoblasts were transplanted, the fibers they formed were numerous, widely dispersed within the limb musculature, and persisted in the muscles until at least ED10. These results indicate that transplanted myoblasts of embryonic, fetal, and adult origin are capable of forming fibers during early limb muscle formation. They also indicate that even in an embryonic chick limb where proliferation of endogenous myoblasts and muscle fiber formation is rapidly progressing, myoblasts that are cultured in vitro do not substantially contribute to long-term muscle fiber formation after they are transplanted into developing limbs. However, when the same myoblasts are freshly isolated and transplanted without prior cell culture, substantial numbers of fibers form and persist after transplantation into developing limbs. Thus, these studies demonstrate that the extent to which transplanted myoblasts fuse to form fibers which persist in host musculature depends upon whether donor myoblasts are freshly isolated or maintained in vitro prior to injection.     

Mechanical function of muscle reinnervated by end-to-side neurorrhaphy.

End-to-side neurorrhaphy is a surgical technique for peripheral nerve reconstruction when end-to-end neurorrhaphy is not an option. To define the effectiveness of end-to-side neurorrhaphy as a method of nerve repair, the authors tested the null hypothesis: there is no difference in the mechanical function of skeletal muscle denervated and reinnervated by end-to-side versus end-to-end neurorrhaphy. Adult Lewis rats underwent either transection and end-to-end epineurial repair of the left peroneal nerve (n = 9) or end-to-side repair of the distal stump of the peroneal nerve to the side of the tibial nerve (n = 8). After a 6-month recovery period, isometric force (Fo) was measured, and specific force (sFo) was calculated for the extensor digitorum longus muscle of each animal. Immunohistochemical staining for neural cell adhesion molecule (NCAM) was performed to identify populations of denervated muscle fibers. The mean extensor digitorum longus muscle mass in the end-to-end group (195 +/- 32 g) was significantly greater than that of the end-to-side group (146 +/- 55 g) (p < 0.05). A significantly greater percentage of denervated fibers was identified in the extensor digitorum longus muscles of animals in the end-to-side group (9.4 +/- 3.2 percent) than in those in the end-to-end group (3.8 +/- 1.0 percent) (p < 0.05). Despite a lower muscle mass and a higher percentage of denervated fibers, neither Fo nor sFo was significantly different in the two groups. These data support the null hypothesis that, under appropriate circumstances, there is no difference in the recovery of whole muscle force and specific force production in muscles reinnervated by end-to-side versus end-to-end neurorrhaphy.     

Collateral sprouting occurs following end-to-side neurorrhaphy

Recent evidence supports the use of end-to-side neurorrhaphy for the treatment of certain peripheral nerve disorders. However, the mechanism by which nerves regenerate following this procedure is still unclear. To address this question, the authors designed a new end-to-side coaptation model in rats in which the donor nerves were uninjured. The regenerated axons at the coaptation site were observed directly using fluorescent dye as the neural tracer. The sciatic nerve from adult Wistar rats was transplanted between the left and right median nerves. Fifteen rats were divided into three groups. In group I, the donor (right median) nerve was sutured end to side to the divided grafted nerve using a noninjury technique. In group II, the aponeurosis of the spinal muscles was harvested and the sciatic and right median nerves were coapted end to side noninjuriously by wrapping them in the excised aponeurosis. In group III, a perineurial window was created and a partial neurectomy was carried out at the suture site, after which the sciatic and right median nerves were sutured end to side. Sixty days after the operation, nerve regeneration was evaluated by recording action potentials in the grafted nerve, by performing electromyography in the flexor muscles in the forearm, and by histological examination. The grafted nerves were fixed and sectioned, the number of regenerated nerve fibers was counted, and axonal diameters were measured. Fluorescent dye crystal was used, in conjunction with confocal microscopy, to observe the regenerated axons at the co-aptation site. The results showed that nerve regeneration had occurred in the animals, as determined electrophysiologically and histologically. Both the right and left flexor muscles of the forearm contracted simultaneously as a result of indirect electric stimulation of the grafted nerve, which suggests that the regenerated nerve was physiologically connected with the donor nerve. Nerve fiber counts did not show any differences among groups (p > 0.05), but axonal diameters were significantly greater in group III than in the other two groups. Fluorescent dye staining revealed the presence of regenerated nerve fibers beyond the coaptation site. In group III, the regenerating nerves were observed within the whole section of the coaptation site and collateral sprouting was found to occur even at a site distal to the suture. From these results, the authors conclude that in end-to-side neurorrhaphy, nerve regeneration occurs by collateral sprouting from the donor nerve.     

Sonic hedgehog (SHH) specifies muscle pattern at tissue and cellular chick level, in the chick limb bud.

Development of the musculature in chick limbs involves tissue and cellular patterning. Patterning at the tissue level leads to the precise arrangement of specific muscles; at the cellular level patterning gives rise to the fibre type diversity in muscles. Although the data suggests that the information controlling muscle patterning is localised within the limb mesenchyme and not in the somitic myogenic precursor cells themselves, the mechanisms underlying muscle organisation have still to be elucidated. The anterior-posterior axis of the limb is specified by a group of cells in the posterior region of the limb mesenchyme, called the zone of polarizing activity (ZPA). When polarizing-region cells are grafted to the anterior margin of the bud, they cause mirror-image digit duplications to be produced. The effect of ZPA grafts can be reproduced by application of retinoic acid (RA) beads and by grafting sonic hedgehog (SHH)-expressing cells to the anterior margin of the limb. Although most previous studies have looked at changes of the skeletal patterning, ZPA and RA also affect muscle patterning. In this report, we investigated the role of SHH in tissue and cellular patterning of forearm wing muscles. Ectopic application of a localised source of SHH to the anterior margin of the wing, leading to complete digit duplication, is able to transform anterior forearm muscles into muscles with a posterior identity. Moreover, the ectopic source of SHH induces a mirror image duplication of the normal posterior muscles fibre types in the new posterior muscles. The reorganisation of the slow fibres can be detected before muscle mass cleavage has started; suggesting that the appropriate fibre type arrangement is in place before the splitting process can be observed.     

Activity in torso muscles during relaxed standing

Electromyographic activity of erector spinae, external oblique, and rectus abdominis muscles was studied during relaxed standing compared to lying down. Activity in the forearm extensors and forearm flexors was also studied. Surface electrodes were used. Each of the torso muscles exhibited 0.2 microV of activity and the forearm muscles 0.1 microV while subjects were relaxed and lying down. During quiet standing the erector spinae, external oblique, and rectus abdominis muscles showed a median activity of 1.0 microV, 2.5 microV, and 0.7 microV respectively (for a minimum of ten 10-sec samples per subject). Examination of the integrated records during standing revealed no periods without increased muscle activity in the torso muscles. By contrast, activity in the forearm muscles did not increase during standing. The major superficial muscles of posture in the torso appear to act as guy wires, being continually active during standing. There is no support for hypotheses of passive support for the torso, nor do torso muscles act in either/or fashion; both anterior and posterior muscles are active at once. There is no sign of generally increased muscle tone in all muscles or in extensors; only the postural muscles are continuously active.     

Absence of MyoD Increases Donor Myoblast Migration into Host Muscle

Donor myoblast migration is a major limiting factor in the success of myoblast transfer therapy, a potential treatment for Duchenne muscular dystrophy. A possible strategy to promote the migration of donor myoblasts into host muscle is to enhance their proliferation and delay their fusion, two properties that are major characteristics of myoblasts in regenerating skeletal muscle in MyoD null (-/-) mice. Here we investigate whether the migration of MyoD (-/-) donor myoblasts into host muscle is enhanced in vivo. Sliced muscle grafts from male MyoD (-/-) or normal control (Balb/c) mice were transplanted into the muscles of female normal (Balb/c) host mice. Muscles were sampled at 1, 3, and 12 weeks after grafting, and the fate of male donor myoblasts within female host muscles determined by in situ hybridization with the mouse Y-chromosome-specific Y-1 probe. MyoD (-/-) donor myoblasts migrated into host muscle continuously over 1, 3, and 12 weeks after grafting, in contrast with Balb/c donor myoblasts, whose overall numbers and migratory distances did not increase significantly after 1 week. These results strongly support a role for elevated donor myoblast proliferation and/or their delayed fusion in enhancing migration into host muscle in vivo, and endorse the use of either genetically engineered donor myoblasts, or the administration of exogenous myoblast mitogens to improve donor myoblast migration in myoblast transfer therapy.     

Direct cord implantation in brachial plexus avulsions: revised technique using a single stage combined anterior (first) posterior (second) approach and end-to-side side-to-side grafting neurorrhaphy

Background

The superiority of a single stage combined anterior (first) posterior (second) approach and end-to-side side-to-side grafting neurorrhaphy in direct cord implantation was investigated as to providing adequate exposure to both the cervical cord and the brachial plexus, as to causing less tissue damage and as to being more extensible than current surgical approaches.

Methods

The front and back of the neck, the front and back of the chest up to the midline and the whole affected upper limb were sterilized while the patient was in the lateral position; the patient was next turned into the supine position, the plexus explored anteriorly and the grafts were placed; the patient was then turned again into the lateral position, and a posterior cervical laminectomy was done. The grafts were retrieved posteriorly and side grafted to the anterior cord. Using this approach, 5 patients suffering from complete traumatic brachial plexus palsy, 4 adults and 1 obstetric case were operated upon and followed up for 2 years. 2 were C5,6 ruptures and C7,8T1 avulsions. 3 were C5,6,7,8T1 avulsions. C5,6 ruptures were grafted and all avulsions were cord implanted.

Results

Surgery in complete avulsions led to Grade 4 improvement in shoulder abduction/flexion and elbow flexion. Cocontractions occurred between the lateral deltoid and biceps on active shoulder abduction. No cocontractions occurred after surgery in C5,6 ruptures and C7,8T1 avulsions, muscle power improvement extended into the forearm and hand; pain disappeared.

Limitations include

spontaneous recovery despite MRI appearance of avulsions, fallacies in determining intraoperative avulsions (wrong diagnosis, wrong level); small sample size; no controls rule out superiority of this technique versus other direct cord reimplantation techniques or other neurotization procedures; intra- and interobserver variability in testing muscle power and cocontractions.

Conclusion

Through providing proper exposure to the brachial plexus and to the cervical cord, the single stage combined anterior (first) and posterior (second) approach might stimulate brachial plexus surgeons to go more for direct cord implantation. In this study, it allowed for placing side grafts along an extensive donor recipient area by end-to-side, side-to-side grafting neurorrhaphy and thus improved results.

Level of evidence

Level IV, prospective case series.     

Face lift and hair transplantation as a single procedure.

Frequently, when a face lift procedure is performed, several pieces of healthy scalp are discarded as waste unless a prehairline incision is used. In selected cases, such as patients with hair loss, these pieces of scalp may be used to create micrografts (grafts with 1 to 2 hairs) and minigrafts (grafts with 3 to 4 hairs) and transplanted to the areas of need in the same session. I have found particularly rewarding the combination of face lift and hair transplantation, because patients who need both procedures benefit immensely by doing them together. This way, the pieces of healthy scalp that normally would have gone to waste are recycled. In a preliminary fashion, a strip of retroauricular and occipital scalp that normally would be discarded is harvested from one side and handed to my assistants. Under magnification, they dissect it into micrografts and minigrafts as I do the face lift on that side. When I go to the second side of the face lift, I give them the other strip of scalp; again, as they dissect it into grafts, I continue with the face lift. Usually, we generate about 1000 micrografts and minigrafts from those strips that would have normally been discarded. If I want more grafts, I would (in a preliminary fashion) harvest the donor strip of the size required. As the face lift with or without eyelids is completed, we usually have the grafts ready for insertion. Today, we are able to transplant approximately 1000 grafts in about 1 hour. Therefore, combining the two procedures adds only about an hour to our surgical and anesthesia time.     

Histological and mechanical evaluation of antifreeze peptide (Afp1m) cryopreserved skin grafts post transplantation in a rat model

The objective of this study was to evaluate the use of Afp1m as a cryopreservative agent for skin by examining the transplanted skin histological architecture and mechanical properties following subzero cryopreservation. Thirty four (34) rats with an average weight of 208 ± 31 g (mean ± SD), were used. Twenty four (n = 24) rats were equally divided into four groups: (i) immediate non-cryopreserved skin autografts (onto same site), (ii) immediate non-cryopreserved skin autografts (onto different sites), (iii) skin autografts cryopreserved with glycerol for 72 h and (iv) skin autografts cryopreserved with Afp1m for 72 h at −4 °C. Rounded shaped full-thickness 1.5–2.5 cm in diameter skin was excised from backs of rats for the autograft transplantation. Non-cryopreserved or cryopreserved auto skin graft were positioned onto the wound defects and stitched. Non-transplanted cryopreserved and non-cryopreserved skin strips from other ten rats (n = 10) were allowed for comparative biomechanical test. All skin grafts were subjected to histological and mechanical examinations at the end of day 21. Histological results revealed that tissue architecture especially the epidermal integrity and dermal-epidermal junction of the Afp1m cryopreserved skin grafts exhibited better histological appearance, good preservation of tissue architecture and structural integrity than glycerolized skin. However, there was no significant difference among these groups in other histological criteria. There were no significant differences among the 4 groups in skin graft mechanical properties namely maximum load. In conclusion, Afp1m were found to be able to preserve the microstructure as well as the viability and function of the skin destined for skin transplantation when was kept at −4 °C for 72 h.