The morphometrical analysis on the ultrastructure of A549 cells.

OBJECTIVE: To report the morphometric characteristics of ultrastructure inside A549 cells. METHODS: A549 cells were processed for inverted microscopy and transmission electron microscopy (TEM). Cell images were obtained randomly using inverted microscopy and TEM. The morphometric parameters of ultrastructure were tested using precise morphometric techniques by Image-Pro Plus analysis software. RESULTS: (1) The diameter of A549 cells from inverted microscopy and TEM images was 14.93 µm and 10.59 µm. (2) By defining cell as reference space the volume densities (VV) of nucleus and cytoplasm were about 0.28 and 0.72; the surface densities (SV) of nucleus were 0.19 µm-1. By defining cell nucleus as reference space the VV of nucleoli, euchromatin and heterochromatin were 0.076, 0.72 and 0.20 respectively; the SV of nucleoli was 0.15 µm-1. By defining cytoplasm as reference space the VV of mitochondria, lamellar bodies and lysosomes were 0.046, 0.025 and 0.014; the SV of mitochondria, lamellar bodies and lysosomes were 0.60 µm-1, 0.36 µm-1, and 0.18 µm-1. (3) In individual A549 cell total volume and surface of mitochondria were 61.91 µm³ and 1001.67 µm²; Total volume and surface area of lamellar bodies were 76.82 µm³ and 428.68 µm²; Total volume and surface area of lysosomes were 21.69 µm³ and 212.04 µm². CONCLUSIONS: The morphometric parameters of some ultrastructures within A549 cells were established using precise morphometric techniques by Image-Pro Plus analysis software.

[Mechanism of rat sciatic nerve regeneration induced by human hair keratin].

OBJECTIVE: To evaluate the effect of human hair keratin (HHK) in peripheral nerve repair and explore the mechanism of sciatic nerve regeneration. METHODS: Rat models of sciatic nerve damage was established by creating a 10-mm gap in the sciatic nerve, which was bridged with a HHK implant. Histological examinations of the nerve tissues were performed at different time points after the surgery. RESULTS: During the period from 2 days to 2 weeks following HHK implantation, Schwann cells were found to undergo dedifferentiation and proliferate along the HHK implant. Three weeks after HHK implantation, numerous macrophages and megakaryocytes occurred around the HHK, and a large quantity of regenerated Schwann cells aligned in orderly fashion was seen between the fine filaments of partially degraded HHK, where axons and capillaries were also observed. Six weeks later, massive nerve fibers and capillaries developed around the HHK, and at 9 weeks, the HHK implant was substantially degraded and numerous regenerated nerve fibers occurred characterized by obvious epineurium and perineurium. Till 12 weeks after HHK implantation, HHK was almost completely degraded and replaced by the newly regenerated nerve fibers that had grown across the nerve defect. CONCLUSIONS: HHK is an ideal material for nerve injury repair. Apocytosis plays a key role in the differentiation process of highly differentiated Schwann cells into immature Schwann cells following nerve injury. As a protective mechanism, the axons undergo enclosure and dissociation following injuries, and the intact axons give rise to growth cones that extend fibers of growing buds to competitively bind the one or more Schwann cells, but only one such but finally develops into a complete axon. The nerve fiber barrier membrane is derived from the capillary menchymal stem cells and the outmost vascular barrier membrane. The regeneration of the Schwann cells, axons and the nerve membrane is the result of self-organization through a well synchronized and coordinated mechanism.

[Construction of artificial nerve bridge by three-dimensional culture of interleukin-1beta- activated Schwann cells with human hair keratins].

OBJECTIVE: To culture interleukin-1beta (IL-1beta)-activated Schwann cells (SCs) with human hair keratins (HHKs) for artificial nerve bridge construction. METHODS: SCs purified by primary culture with or without IL-1beta activation were cultured with HHKs decorated by extracellular matrix (ECM), and the artificial nerve bridge was implanted into the defect of rat sciatic nerve. The morphology of the SCs cultured with HHKs was monitored by inverted microscope, scanning electron microscope and evaluated by immunocytochemical staining, and the expression of nerve growth factor (NGF) in the sciatic nerve was observed by in situ hybridization. RESULTS: Activated SCs showed better ability to adhere to the HHKs and grew well. The HHKs component in the artificial nerve bridge underwent degradation in the sciatic nerve defect after 3 to 4 weeks, and IL-1beta activation resulted in enhanced NGF expression in the SCs. CONCLUSION: The constructed artificial nerve bridge by three-dimensional culture of IL-1beta-activiated SCs with HHKs decorated by ECM promotes the repair of sciatic nerve defects and accelerates sciatic nerve regeneration.

Mesenchymal stem cells differentiate into tenocytes by bone morphogenetic protein (BMP) 12 gene transfer.

Rhesus bone marrow mesenchymal stem cells (MSCs) were transfected with the BMP12 gene by electroporation, and the phenotype of the transfected cells was identified by morphological observation and molecular biological assay. After transfection, cells became slender, and their processes became thinner and were interwoven into a network. There were more organelles in the transfected cells than in the parental MSCs. The transfected cells exhibited mRNA expressions of BMP12, collagen type I and scleraxis, but not collagen type III mRNA expression. Immunocytochemical analysis also showed the presence of collagen type I, but not collagen type III in the transfected cells. The transfected cells were positive for CD44 and negative for HLA-DR. Therefore, MSCs can be introduced to differentiate into tenocytes by BMP12 gene transfection, and bone marrow MSCs can serve as an alternative seed cell for application in tendon tissue engineering.

[Construction of artificial nerve bridge by three-dimensional culture of Schwann cells with human hair keratins].

OBJECTIVE: To culture Schwann cells (SCs) and human hair keratins (HHKs) for artificial nerve bridge construction. METHODS: SCs were purified by primary culture and labeled with BrdU, which were then cultured with HHKs decorated by ECM. The artificial nerve bridge was implanted into the defect of sciatic nerve, beneath the skin, and in the skeletal muscles of SD rat, respectively. The morphology of the SCs cultured with HHKs was monitored by inverted microscope and evaluated by immunocytochemical staining. Growth of BrdU-labeled SCs in vivo was observed by immunocytochemical staining on paraffin sections. RESULTS: In vitro cultured SCs were capable of adhering to HHKs and grew well four weeks after implantation. The HHK component in the artificial nerve bridge underwent degradation in the defect of the sciatic nerve, beneath the skin, and in the skeletal muscles of SD rat, and SC survival and proliferation were verified. CONCLUSION: SCs can survive in three-dimensional culture with HHKs for construction of artificial nerve bridge to repair nerve defects.

[FK506 promotes apoptosis of macrophages activated by homogenate of allogenic nerve].

OBJECTIVE: To study the effect of FK506 in promoting apoptosis of peripheral blood-derived macrophages activated by homogenate of allogenic nerve tissues. METHODS: Homogenate of the allogenic nerve tissues was prepared using the sciatic nerve and injected in one-month-old SD rats, from which the macrophages activated by the homogenate were collected from the abdominal cavity and cultured in vitro. The cells were divided into 4 groups according to different concentrations of FK506 for treatment, namely 0 (group A, control group), 0.25 ng/ml (group B), 0.5 ng/ml (group C), and 1.0 ng/ml (group D). The cells of the 4 groups were inoculated into 96-well plate respectively for detecting the viability of the macrophages by MTT assay and for morphological evaluation of the cell apoptosis by transmission electron microscopy and fluorescence microscopy. RESULTS: The cells in groups B and C exhibited reduced viability and signs of apoptosis, and necrosis was observed in group D. Transmission electron microscopy and fluorescence microscopy identified early apoptotic changes and the presence of apoptotic body in the macrophages. The apoptotic rates of groups B and C were much higher than that in group A found by flow cytometry. CONCLUSION: FK506 can promote the apoptosis of macrophage activated by allogenic nerve homogenate and reduce macrophage-mediated immunological rejection of peripheral nerve allograft.

[A new method for screening mutant yeast strains with green fluorescent protein].

OBJECTIVE: To establish a fast and simple method for screening mutant yeast strains. MATERIALS AND METHODS: Homologous recombination technique was used to detect mutant yeast strains in yeast genomic library, with the green fluorescent protein gene as the reporter gene in the transposon. RESULTS: The strains that emitted green fluorescence were isolated, indicating that the gfp gene was inserted into the yeast genome by homologous recombination. CONCLUSION: This study established a useful method for functional genome study by homologous recombination technique, and provide an alternative for gene therapeutic drug development.

[In vivo tissue engineering: a new concept].

From our over a decade-long experience in experiments and clinical applications of human hair keratin (HHK) artificial tendon, a conclusion was drawn that HHK artificial tendon components and their degradation products could stimulate the proliferation and differentiation of tenoblasts in the neighboring tissues into tenocytes. With the regulation of this process by certain cytokines secreted by other cells, autotendons can be finally formed. We also found that after grafting for the tissue defects such as in the bone, nerve and muscle, "in situ construction" of the tissue/organ substitutes occurred, which inspired us to propose a wholly new theoretical system--in vivo tissue engineering, defined as in vivo reconstruction of the defected tissues or organs. The grafted absorbable scaffold biomaterial itself and its degradation products can activate the mitosis, proliferation, and differentiation of adult stem cells in the surrounding tissues, which organically interact with the material to form an organic complex under in vivo physiological conditions. Finally the matrix material is completely replaced by the complex, an almost identical structure with the normal tissue in terms of anatomy and histology. One of the advantages of in vivo tissue engineering is "in vivo construction" of the tissue/organ substitutes in anatomy, histology and function way of "in vivo cultivation" of the seed cells under in vivo microenvironment and in vivo precise regulation mechanism. It solves such problems as immune rejection against seed cells, variation and functional deterioration of the seed cells, complicated preservation procedure, transportation and high cost. The most prominent advantage of this technique is that it best meets the clinical need, not only in the sense of its powerful clinical potential, but also in its significant theoretical value.

[Dedifferentiation and regeneration of damaged cells and tissues].

Dedifferentiation is an important event in developmental biology and morphology but frequently neglected by the biologists. In the past, attention was given predominantly to its relationship with tumorigenesis. In our series of researches, we found important and inherent relations between dedifferentiation and regeneration of damaged cells and tissues in the emergence of large numbers of Schwann cells that digested their own myelin sheath components and senescent organelles by autophagic mechanism during regeneration of the peripheral nerves following injuries, when some apocytosomes (apo+cyto+sis), by means of apocytosis (apo+cyto+sis), budded from the cells containing such primitive organelles as free ribosomes and polyribosomes and retaining a very small quantity of active mitochondria and Golgi apparatus, all characterizing an immature state of the cells after dedifferentiation. Many autophagic bodies were found enwrapped by membranes L02 cells (a liver cell line) after vincristine-induced damage, with numerous apocytosomes dissociated following budding around the cells. The cells survived the treatment presented immature appearance, a phenomenon we termed as dedifferentiation, after which the metabolic burden of the cells was relieved, the senescent and damaged organelles were cleared, and the cells resumed their viability and proliferated vigorously to repair and reconstruct the damaged tissues. As most researchers have currently give their full attention to the important role of stem cells in the regeneration of damaged cells and tissues, we, after long-term observations and experiments, propose that mature cells also take part in the regeneration and reconstruction process after dedifferentiation.

Autophagy of neuron axon during regeneration of rat sciatic nerves.

OBJECTIVE: To investigate the autophagic clearance of degenerated neuron axon during regeneration of rat sciatic nerves. METHODS: Wallerian degeneration model was established in rats by sciatic nerve transection. Samples from the distal stump were collected at different time points after the transaction and ultrathin sections prepared for electron microscopic examination and acid phosphatase (AcPase) activity detection. RESULTS: Neuron axon degeneration occurred after transection of rat sciatic nerve, presenting predominantly swelling of the axoplasm and separation of the axon from the myelin sheath seen 5 h to 2 d after the transection. On day 4, axoplasm condensation took place and the axons were completely separated from the myelin sheath to form dissociative axon body. Vacuoles of various sizes were identified in the axon in the early stage after operation and later when the axons were completely dissociated from the nerve sheath, larger dissociative axon bodies occurred. The axolemma surrounding the axon body was derived from the neuronal cytomembrane, and the condensed axoplasm contained numerous autophagic vacuoles at all levels along with large number of neurofilaments, microtubules and microfilaments arranged in a crisscross pattern. The autophagic vacuoles exhibited acid phosphatase (AcPase) activities. Since the day 7, the axon bodies were absorbed after degradation and macrophages could be spotted occasionally. CONCLUSION: The degenerated axons were cleared mainly through autophagy during regeneration of rat sciatic nerve and macrophages only assist in this process.

[Cloning and sequence analysis of the preprotein cDNA of human bone morphogenetic protein 12].

OBJECTIVE: To clone the cDNA of the preprotein cDNA of human bone morphogenetic protein 12 (hBMP12). METHODS: Two primers were designed according to hBMP12 sequence reported in GenBank. The hBMP12 preprotein cDNA was obtained by reverse transcriptional (RT)-PCR from the mRNA extracted from human placenta, followed by cloning into pTARGETTM plasmid and sequence analysis of the plasmid pT(ARGE)T(TM)-BMP12. RESULTS: DNA agarose gel electrophoresis showed that the product of RT-PCR was about 920 bp, as was consistent with the result of PCR detection of the recombinant plasmid. The result of sequence analysis was in agreement with the reported hBMP12 sequence in GenBank. CONCLUSION: The preprotein of hBMP12 cDNA has been successfully cloned with correct sequence.

[Autophagic effect of Schwann cells in the regeneration of rat sciatic nerves].

OBJECTIVE: To investigate the autophagic effect of Schwann cells in the process of rat sciatic nerve regeneration. METHODS: Wallerian degeneration model was established by transecting the rat sciatic nerve. Samples from the distal stump were obtained 0, 0.5, 1, 1.5, 2, 3, 4, 5, 7, 10, and 15 days respectively after the transaction, and ultrathin sections were prepared for examination with electron microscope. RESULTS: Axons started to separate from the myelin sheath at day 0.5 after the transection, followed by rapid vacuolar degeneration. Since day 2, the myelin sheath folded and broke into fragments, and in the Schwann cells large cell membrane-bound myelin debris and many scattered small fragments could be seen to fuse with the lysosomes to form ACPase reaction-positive autophagic vacuoles. Immature cells were occasionally seen in the endoneural space, appearing in large amount a week later. After day 7, the number of autophagic vacuoles began to diminish. In the entire course of the observation, macrophages containing autophagic vacuoles, were spotted only occasionally. CONCLUSION: The degenerated myelin debris are cleared mainly through the mechanism of autophagy by the Schwann cells during regeneration of rat sciatic nerve. Schwann cells dedifferentiate into Schwann cell precursors, which then proliferate and differentiate to take part in the regeneration of the nerves.

[Association of receptor-mediated endocytosis and autophagy with apoptosis].

OBJECTIVE: To evaluate the relationship between receptor-mediated endocytosis, autophagosome formation and apoptosis by observing the morphological changes of mouse peritoneal macrophages in the course of receptor-mediated endocytotic activities, autophagy and apoptosis. METHODS: Mouse peritoneal macrophages were incubated with horseradish peroxidase-labeled concanavalin A (ConA-HRP), and morphological examinations were performed at different time points after the incubation. RESULTS: After the incubation of the macrophages with ConA-HRP, ConA-HRP was observed to enter the vesicles by way of receptor-mediated endocytosis. Three kinds of endosomes were observed, namely vesicles, tubes and double-membrane sheets. The double-membrane sheets enveloped a portion of the cytoplasm and organelles, thus giving rise to vesicles, or the autophagosomes, which later fused with lysosome, followed by the apoptosis of the macrophages. CONCLUSION: Receptor-mediated endocytosis of ConA-HRP is associated with autophagy and apoptosis.

[Expression and distribution of NGF and p75 during rabbit tibial nerve repair induced by human hair keratin conduits].

OBJECTIVE: To study the expression and distribution of nerve growth factor (NGF) and low-affinity neurophin receptor p75 during human hair keratin conduit-induced repair of rabbit tibial nerves. METHODS: Rabbit tibial nerves were transected and connected by either routine suture or by conduits made of human hair keratin (HHK), and after different time periods, paraffin-embedded sections of the nerve tissue at the damaged sites and the adjacent tissues, with normal rabbit tibial nerve sections as control, were prepared for immunohistochemistry. RESULTS: No positive NGF staining was observed in normal tibial nerve tissues, but 76 days after the surgery, strong NGF positivity was detected in the newly generated nerve tissue around the HHK implants until 100 days after the surgery, which was absent in the tissues around the suture. As for p75, there was no positive staining observed in normal tibial nerve tissue. Light positive p75 staining was found in the mature nerve tissues around the HHK implants, where the newly generated tissues were strongly p75-positive during the period between 76-and loo-days after surgery. CONCLUSIONS: HHK and its degenerative product, but not routine suture, can induce the production of NGF and p75 to create a favorable micro-environment for nerve regeneration. More NGF and p75 are produced in newly generated neurons than in mature ones.

Effects of resveratrol on secondary damages after acute spinal cord injury in rats.

AIM: To study the effects of resveratrol (Res) on secondary spinal cord edema, the activity of lactate dehydrogenase (LDH), Na+, K+-ATPase, and malondialdehyde (MDA) content in experimental spinal cord injured (SCI) rats. METHODS: The weight-dropping method was used to produce the experimental SCI in adult rats. Res ( 50, 100 mg/kg) and methylprednisolone (MPSS) 100 mg/kg were injected ip immediately after the induction of SCI. The effects of Res on edema, LDH, Na+, K+-ATPase, and MDA were determined at 1 h, 24 h, and 48 h after SCI compared with MPSS. The electron microscope was employed to investigate the ultrastructural effects of Res on axons, neurons, and subcellular organelles after SCI. RESULTS: Res obviously inhibited the secondary spinal cord edema with the most remarkable suppressing rate by 11.5 % at 48 h. Res significantly suppressed the activities of the lactate dehydrogenase with the highest suppressing rate > 40 % at 24 h. Res markedly improved the Na+, K+-ATPase activities that were promoted to the biggest extent of 60 % at 48 h. At the same time, Res (50 and 100 mg/kg) obviously reduced MDA production in the injured spinal cord tissue in comparison with the SCI model, the most remarkable effect of Res was detected at 48 h with the inhibitory rate >40 %. The ultrastructural findings suggested that SCI caused profound spinal cord damage, which could be protected or improved by injection of Res and MPSS. CONCLUSION: Both Res and MPSS effectively protected the spinal cord from secondary spinal cord injures. But the effects of Res 50 and 100 mg/kg were stronger in improving the energy metabolism system and inhibiting the lipid peroxidation in the local injured spinal cord after SCI than MPSS at the dose of 100 mg/kg. Res might have greatly potent therapeutic effects on SCI.

[Effect of human hair keratin implant on oligodendrocyte proliferation and differentiation in rats with acute spinal cord injury].

OBJECTIVE: To investigate the changes of oligodendrocyte proliferation and differentiation in response to implantation of degradable human hair keratin (HHK) into the spinal cord with acute injury in adult rats. METHOD: Rat models of acute spinal cord injury were established by direct impact of the exposed spinal cord with a weight-dropping device, 12 of which received immediate HHK implant into the injured spinal cord. The rats of control injury group (n=12) were subjected to the injury but did not receive subsequent implant, and those in sham operation group (n=12) only had the spinal cord exposure without injury. Another 8 rats were used as the normal control group. Samples of the spinal cord were obtained 1, 4, 12, and 26 weeks respectively after the operations and serial sections were prepared for examination with light and electron microscope. RESULTS: One week after the injury, few oligodendrocytes were observed among the large number of the infiltrating leukocytes in the injured spinal cord with HHK implants. Staining with hematoxylin eosin and Mallory's phosphotungstic acid-hematoxylin at the fourth week revealed oligodendrocyte proliferation around the HHK implant. The period from the 12th to 26th week was characterized by nerve regeneration and myelin sheath reconstruction, in the course of which empty cavity occurred within the sheath of the oligodendrocytes, and lamellar separation of the myelin sheath were observed. Phagocytized myelin sheath and axone fragment were detected in oligodendrocytes, with the newly generated oligodendrocytes scattering abound the rebuilt myelin sheath. CONCLUSION: HHK can be beneficial for the proliferation and differentiation of oligodendrocytes and myelin sheath rebuilding and repair in the course of neuronal regeneration after acute spinal cord injury.

[Morphological changes of collagen fibrils during the formation of autogenous tendon induced by human hair keratin artificial tendon].

OBJECTIVE: To observe the morphological changes of collagen fibrils during the formation of autogenous tendon induced by human hair keratin (HHK) artificial tendon. METHODS: Rabbit models of injured tendon were established in which implantation of HHK artificial tendon was performed to observe the formation of autogenous tendon under light microscope and electron microscope at 3, 6, 9, 12 and 16 weeks after HHK implantation. RESULTS AND CONCLUSION: During autogenous tendon formation induced by HHK artificial tendon, the tendon cells of the impaired end of the tendon and beneath the tendon membrane dedifferentiate and are capable of collagen secretion, followed by the formation of typesI,II and III collagen fibrils, and eventually, the majority of the tendon cells disappear with the collagenization of the tendon.

[Ultrastructural observation of rat thymus tissue with coronavirus infection].

OBJECTIVE: To investigate the ultrastructure of rat thymus tissues with rat coronavirus (RCV) infection for clarifying the mechanism responsible for the morphological changes of the cells infected by RCV. METHODS: Routine electron microscopy was performed for observing RCV-infected rat thymus tissues. RESULTS: Following RCV infection, endoplasmic reticulum (ER) pools of different dimensions were observed in the cytoplasm of the thymic epithelial reticular cells, merging subsequently with each other into larger ER lakes filled with particles of mature RCV, or viral inclusion bodies. After germination on the ER membrane, the viruses entered the matrix of the ER lake to mature and were eventually excreted to the extracellular space. The RCV particles were spherical in shape with a diameter of 100-130 nm and two distinct membranes, the outer one being the envelope and the inner one the nuclear capsid to enclose the viroplasm. Between the envelop and nuclear capsid was a electron-lucent middle layer comprising one to two thin membranous structures. Large quantity of short spike-like projections starting from the nucleus capsid penetrated the middle layer and the envelop to reach the glycoprotein coat and formed a corona-like structure. Mature RCV particles were distributed around the ER pools, cytoplasm, and intercellular space, and the RCVs in the endosome/lysosome were devoid of the envelop and nuclear capsid. CONCLUSION: The ER lakes are involved in the maturation of the viruses, and the envelop and nuclear capsid of the virus entering the cells from extracellular space are removed and degraded in the endosome/lysosome. Replications of virus occurs in plasma of the thymic epithelial reticular cells, and no RCV can be detected in the thymocytes.

Study of direct lung injury by seawater in canine models.

OBJECTIVE: To study the mechanism of direct lung injury by seawater and explore its possible management. METHODS: To exclude the interference of hypoxia and acidosis during the study of seawater-induced direct lung injury, 18 normal hybrid dogs were randomly assigned into group A (with all lung lobes perfused with seawater), group R (with the right lung lobe perfused with seawater) and group D (with the diaphragmatic lobe of lung perfused with seawater), with 6 dogs in each group. The changes in blood gas dynamics, blood gas acid-base status and electrolytes, along with the histological changes in the lung tissues were comparatively analyzed between the 3 groups. Bronchial microscope was employed to observe the continuous changes in the bronchioles before and after seawater perfusion in group D, and the concentration of the bronchoalveolar fluid and blood LDH-L and ALP levels were tested. RESULTS: The values of PaO(2), PaCO(2), pH, actual bicarbonate (AB), base excess (BE), tidal volume, and respiration rate in groups A and R were significantly different from those in group D (P < 0.01), and in groups A and R, the above measurements at every stage after seawater perfusion were significantly different from those before perfusion (P < 0.01). In group D, however, blood gas dynamics, blood gas acid-base status and electrolytes changed little after seawater perfusion (P > 0.05). In all the groups, obvious lung tissue injuries were observed under optical microscope after seawater perfusion. Observation with electron microscope revealed injuries to type II alveolar epithelial cells, broadened respiratory mucosa, and platelet adherence. Bronchial microscope in group D presented the bronchus filled with bronchoalveolar fluid, and blood LDH-L and ALP levels kept rising significantly (P < 0.01). Within 4 h after seawater perfusion, no pathological changes were seen in the lung tissues without direct contact with seawater. CONCLUSIONS: Seawater inspiration and retention in the lungs may lead to severe direct lung injury, and is the primary factor responsible for acute lung injury after drowning in the sea.

Degradation of human hair keratin scaffold implanted for repairing injured skeletal muscles.

OBJECTIVE: To observe the in vivo degradation process of human hair keratin (HHK) scaffold after implantation in rabbits. METHODS: Seven New Zealand rabbits were divided into 4 groups including a control group and 3 operation groups. HHK scaffold was implanted, after partial resection of the skeletal muscles, in rabbits of the 3 operation groups, followed by observation 1, 3, and 6 weeks later respectively. Routine morphological observation, histochemistry with ubiquitin and electron microscopy were performed. HHK scaffold incorporated 3 types of HHK with different degradation speeds, respectively designated types F, B, and Z. RESULTS: Light microscopic observation revealed that human hair cuticles began to strip off at the first postoperative week, and the material was homogeneous on the surface of which macrophagocytes and multinuclear giant cells adhered. At the third week HHK scaffold was degraded into particles as seen under electron microscope and was phagocytosed by macrophagocytes and multinuclear giant cells. Ubiquitin enzymatic histochemistry demonstrated that macrophagocytes, multinuclear giant cells were positive at the first week. At sixth week, further degradation of HHK scaffold occurred when newly generated muscles were seen beside the HHK. CONCLUSIONS: HHK scaffold is initially degraded extracellularly by ubiquitin system into particles, which are phagocytosed by the cells and degraded by the cooperation of lysosome and ubiquitin; meanwhile the satellite cells are activated, beginning to proliferate and eventually fused into newly generated muscle fibers.