Acellular Research Articles

Experimental study of compatibility between chondrocytes and allogenic cartilage microparticle acellular tissue matris in vitro

To construct tissue engineered cartilage using cartilage microparticle acellular tissue matrix (CMACTM) as scaffold. To determine the content of hydroxyproline, glycosaminoglycan and DNA of CMACTM prepared from sheep's articular cartilage with multistep enzymic method, and to analyze CMACTM with gross observation, histology and scanning electron microscopy. Allogenic chondrocytes were mixed with CMACTM and cultured in vitro from 0 to 35 days. Observations through inverted microscope, scanning and transmission electron microscope, quantifications of hydroxyproline, glycosaminoglycan and DNA in the composite, cells adhesion rate were applied to analyze the results. The diameter of CMACTM was 0.100-0.154 mm, which contain extracellular matrix only. Hydroxyproline, glycosaminoglycan and DNA quantifications in CMACTM were 204.374 +/- 3.120 microg/mg, 18.302 +/- 2.037 microg/mg and 0.042 +/- 0.013 microg/mg respectively. Allogenic chondrocytes enclosed CMACTM tightly, hydroxyproline, glycosaminoglycan and DNA quantifications in the composite of the two formers increased with difference on 7th day compared with that on 0 day, reached to the peaks on 14th day (hydroxyproline, DNA) and on 21st day (glycosaminoglycan), and retained at a high level on the following days. Cells adhesion rate was 92%. Allogenic CMACTM possessed satisfactory biocompatibility for chondrocytes and provided a new scaffold for cartilage tissue engineering.

The eyelid reconstruction in situ with allograft acellular dermal matrix

To explore the clinical value of allograft acellular dermal matrix in the treatment of eyelid defects. From November 2001 to November 2002, 10 patients (8 male and 2 female; age varied from 14 to 47) with 11 eyelids were treated by eyelid reconstruction in situ with implantation of 1 mm thick allograft acellular dermal matrix. In 11 eyelids with defects, 2 was totally defect, 8 was 2/3 defect, 1 was 1/3 defect. In these 10 patients, 9 were injured and 1 was congenital. These defective eyelids included 8 upper lids, 1 lower lid and 1 case with both upper and lower lids. The average follow-up time was 8 months. In the follow-up periods, the inflammation of the eyelid was slight and no implants shedding or infection happened postoperatively. The conjunctiva grew well on the surface of the acellular dermal matrix. Histology examination showed neovascularization and presence of fibroblasts in the acellular dermal matrix 6 months after the operation. By the end of the follow up period, the results were excellent in 8 eyelids, good in 2 eyelids and improved in 1 eyelid. The efficacy rate of this treatment was 91%. Acellular dermal matrix can be used as a substitute of the tarsus in supporting the eyelid, it invokes little immunological and inflammatory reaction, and therefore, it can act as a biological scaffold to guide the growth of collagen and neovascularization.

Homologous dermal acellular matrix graft for urethral reconstruction in man (report of 16 cases)

To investigated the value of homologous dermal acellular matrix graft (AMG) for urethral reconstruction in man, and found an ideal replacement material to simplify the urethroplastic surgery. 16 male patients, aged 18-46 (mean 26.5), with urethral diseases were treated with allograft of tissue engineering material AMG. The causes of urethroplasty were complex urethral stricture followed by pelvic fracture (13 cases), anterior urethral stricture (2 cases), hypospadia (1 case). During the operation, AMG was sutured to a tubular graft and replaced the defect urethra. A 18-22 Fr silicone catheter was inserted in the reconstructed urethra for 4-6 weeks. All 16 patients were voiding well after removing the catheter, urethrography reveal excellent caliber of the reconstructed urethra. Urethroscopic examination showed the graft urethra was covered by epithelial tissue and grew into the native tissue. But stenosis was developed in 3 patients postoperatively, 1 patients received transurethral incision, another 2 needed urethral sounding. The patients were followed-up for 12-72 months (mean 45.6), no rejection was observed, all patients voided normally except 4 cases needed urethral dilatation periodically. The homologous dermal acellular matrix graft may serve as an ideal replacement material for complex urethral stricture or defect, without concern of rejection.

Effects of IGF-1 and bFGF on the ECM synthesization and cell proliferation by cultured rabbits auricular elastic chondrocytes in alginate beads

Objective To investigate effects of insulin-like growth factor and basic fibrobla st growth factor on the cell proliferation and synthesized extr acellular matrix of cultured chondr ocytes.Methods Isolated chondrocytes from rabbit auricular cartilage by enzyme digestion were encapsulated in alginate i n a density of 10 7 cells /ml.The alginate beads of chondrocytes were prepared by dropping the cells /alginate into 125mM Ca Cl2solution through a 22gauge needl e and were allowed to polymerized for 10min.The beads were cultured for 14days in vitro with DMEM added with 10%FBS for bio-chemical analysis.In groups A,B and C,25ng /ml IGF-1,50ng /ml bFGF,25ng/ml IGF-1and 50ng /ml bFGF were added respectively in the same culture med ium.Results14days later,each beads contained a n average of 3.5μg,3.6μg,6.2μg and 6.6μg DNA.There was no statistical signi ficance between group A and control beads,and the same result was in group B and C.However,the amount of D NA demonstrated a significant incre ase in group C compared to group A and c ontrol.The amount of GAG were 15.5μg,23.8μg,29.0μg and 38.9μg.It was statistically significant in all groups.Conclusions This study demonstrates that IGF-1a nd bFGF influence differently on the ECM synthesization and cell prolife ration of elastic chondrocytes.IGF-1increases the a mount of GAG,but has no significant i nfluence on the cell proliferation.In reverse,the effect of bFGF is on the cell proliferation primarily.When incubated in c ulture medium with both 2growth factors,the chon-drocytes show increasingly prolife ration ability and GAG synthesizati on.It suggests that the combination of different growth factors has an advantageous effect o n the chondrogenesis.[

Risk of Brain Damage Following Pertussis Immunization with Whole-Cell cf Acellular Vaccines

Serious neurological disorders reported following whole-cell (WC) in comparison to acellular (AC) pertussis vaccines (PV) were evaluated by the Genetic Centers of America, Silver Spring, MD.

Nerve regeneration and functional recovery after a sciatic nerve gap repaired by an acellular nerve allograft made through chemical extraction

Objective To observe the nerve regeneration and functional recov er y in canines, in which a large gap was made in the continuity of the sciatic ner ve and the gap was repaired by acellular allograft through chemical extraction. Methods 15 canines were divided into acellular nerve allografting group (ANG, 6 canines), autografting group (AG, 6 canines) and fresh nerve allografting group (FNG, 3 canines). The sciatic nerves on the right side of all animals were expos ed and 5.0 cm long segments were removed from the mid-thigh level and replaced by one of the three types of grafts. The motor recovery was assessed by gait tes t at month 6 postoperatively. The sensory and motor conducting functions were as sessed by electrophysiological experiments. The nerve regeneration was revealed by morphological studies on the grafting segments, the distal tibial nerves and motor end-plate of the target muscle. Results All the animals in ANG and AG gro ups had similar patterns of right posterior limb gait cycle and right ankle move ments. Stimuli (1.0-2.0 mA, 0.1 ms, 1.0 Hz) to the sciatic nerves proximal to t he implanted segment resulted in motor-evoked potentials recorded from the musc ulus triceps surae. The motor conducting velocities of the grafting segments wer e on average 47.2 m/s in ANG, 60.9 m/s in AG and 122.0m/s in normal controls. St imuli (5.0-10.0 mA, 0.2 ms, 1.9 Hz) to the right distal tibial nerves resulted in sensory-evoked potentials recorded from the cortical area in all the canines in ANG and AG groups. Axons regeneration and Schwann cells migration had reoccu pied the empty basal lamina tubes of the acellular allografting segments without excessive sign of inflammation. ANG and AG had similar nerve regeneration with massive large nerve fibers in the distal tibial nerves and vast motor end-plate in the target muscle. Conclusions The patterns of functional recovery and nerve regeneration tend to be similar 6 months after implantation in the canines, in which a 5.0 cm gap in the continuity of the sciatic nerve is repaired by either chemical acellular nerve allografting or autografting.

The efficacy of whole cell pertussis immunisation: collected data on a vaccine produced in France.

The place of whole cell pertussis vaccines in paediatric immunisation schedules is under re-evaluation by public health authorities in many countries, with the expectation that the newly licensed acellular Bordetella pertussis vaccines will induce fewer adverse events while providing equivalent efficacy. In France, for instance, the CSHPF (Conseil supérieur d'hygiène public de France) recently modified its long-standing recommendation that French children only receive whole cell pertussis vaccine. Consequently, an acellular pertussis vaccine may be used for the first booster, at 16-18 months of age, and should be used for the reinforcing dose at 11-13 y of age. French children, nonetheless, continue to receive whole cell pertussis vaccine for the primary series immunisations at 2, 3, and 4 months, as the only whole cell pertussis vaccine available in France (licensed by Aventis Pasteur) has a long-established record of safety and protective efficacy. A review of its unpublished and published clinical results, obtained from studies throughout the world, demonstrates an efficacy of from 84-100% in six different retrospective analyses or outbreak investigations and a protective efficacy of 92% by clinical trial.

Advances of Wound Coverage - Dermal Template Transplantation

The function and appearance of grafted skin over the recipient wound is depended upon the thickness of the donor skin. In extensive burn injury, the donor skin is not always available in the acute stage and usually patient needs a temporary skin substitute to cover the wound, the better long enough until the surgeon has chances to reharvest the same donor site. To use very thin skin for grafting can ensure successful skin graft take and permit faster donor site healing but leave recipient wound contraction. The harvest thick skin for grafting will produce deep wound over donor site and induce infection, scarring, blistering, and pigmentation. During the wound coverage, if we can support additional dermal template other than the autologous dermis may promote prompt and perfect quality healing for recipient wound. In 1995 Alloderm (Life Cell Inc., the Woodland, Texas) was successfully applied to cover full-thickness burn wound with applied a piece of meshed thin split-thickness skin autograft. Since then, the Alloderm had been utilized as a substitute to cover full-thickness burn wound, release scar, repair the nasal septum and for lip agumentation. Our personal experiences also confirm with same result except the meshed skin mark is always unacceptable by most of the patients. Since 1980, Yannas and Burke began to design a bilayer artificial skin, finally named as ‘Integra’, composed of a temporary silastic epidermis and a porous collagen-chondroitin 6-sulfate fibrillar dermis and applied it to cover extensive burn wound. The neodermis was then covered by a piece of ultra thin split-thickness skin after the silastic epidermis was removed. In 1988, Heimbach had reported a multi-center clinical trial by using Integra to cover extensive burn wound and showed that the Integra could provide a vascularized dermal template to take ultra thin split-thickness autologous skin graft. Their long term follow up study also showed that the grafted wound demonstrated minimal scar contraction with rare complications. In 1998, Pandya had provided that Integra can provide a stable environment to support cultured epidermal keratinocytes growth for 1 year firmly (18). Decontaminated pig skin had been used as a biological dressing for burn wound coverage since 1966. In 1984, the porcine dermis had been utilized as a dura substitute, urinary bladder sling, to repair orbit defects and inguinal hernias with acceptable biocompatibility. In 1995, Livesey investigated the potential use of acellular porcine dermis as an allograft combine with a meshed split-thickness autograft can successfully to cover a full-thickness skin wounds in pig model. In 1995 we used meshed porcine dermis combined with a widely expanded mesh autologous thin split-thickness skin (ASTS) graft for a rat wound. Wounds were studied by clinical inspection for texture and contraction, and by histological and immuno-fluorescent techniques. The results showed that the integrity of this complex skin graft was maintained for 4 months with acceptable wound contraction. The cultured epidermal autograft (CEA) was first described in 1984 for cover extensive bum wounds. Unfortunately the CEA showed poor adhesion to recipient wound due to inadequate formation of rete ridges between neoepidermis and wound bed. Evidences had indicated that the formation of rete ridge from CEA needs a recipient wound with dermal-like matrix, such as collagen-glycosaminoglycan (C-GAG), allodermis, autodermis or, must apply fibrin glue to create tight interface integration. In the recent 20 years, because of shortage of the allogenic skin substitute, we had produced 4 generations of pig skin to help for high quality wound healing, they are: 1. Regular porcine skin, 12-15/1000 inches, epidermis / dermis, for temporary wound coverage. It is usually be removed or sloughed by 7-10 days after tansplantation.2. Acellular porcine dermis, 20/1000 inches, when transplantation, it usually combined with a sheet of autologous thin skin overlay. It is a permanent graft.3. Silicon-Acellular Porcine Dermis (SAPD), need 2-stage procedures a. Thin SAPD - temporary / 4-6 weeksb. Thick SAPD - permanent, need autologous STSG / CEA for wound coverage in the second procedure at day 10-14. The safety of using the porcine products had been discussed in the past. In 1993, there is an occasion to developed lymphocytic meningitis with culture negative y using of a porcine dermis implant to repair an operative dura defect, which was considered a hypersensitivity reaction to the porcine dermal implant. The existence of the porcine endogenous retorovirus (PERV) DNA during transplantation of porcine products had been always be raised, and will be discussed.

The Longevity of AlloDerm vs. Zyplast

Because of the limited longevity of Zyplast and the risk for allergic reactions with its use, alternate filler materials are under development. AlloDerm is a cadaver-derived, acellular dermal graft material for soft-tissue augmentation, available as sheets or in injectable form. In this bilateral comparison study, 10 subjects had 0.5 ml of Zyplast implanted intradermally …

Acellular Dermal Graft for Soft Tissue Augmentation

Tissue augmentation with bovine collagen (Zyderm, Zyplast) is effective, but some patients develop allergic reactions, and the soft-tissue-defect

The use of composite acellular allodermis-ultrathin autograft on joint area in major burn patients--one year follow-up.

Scar contracture is a major long-term sequelae of meshed split-thickness skin grafts in the case of full-thickness burn injuries, and especially in joint areas. Severe contracture around joints may lead to loss of mobility. The thickness of the dermis in the autograft plays a major role in the functional and cosmetic outcomes of the third degree burn. How to replace lost dermis is a major problem in skin restoration following severe burns. The human allograft skin (AlloDerm) is an acellular dermal matrix with normal collagen bundling organization and intact basement membrane complex. We present 12 cases of clinical application of a composite grafting technique in which the allogeneic skin (AlloDerm) provided source of dermis, and an ultrathin autograft (0.004-0.006 inch in thickness) provided epidermis. In these patients, The composite grafts were applied to full-thickness burn wounds over various articular skin surfaces. The average skin graft take rate was 91.5%. These ultrathin autografts allow the donor sites to heal faster. The mean time of donor site reepithelization was 6 days. All patients had a nearly normal range of joint motion (average 95% of normal) after one year's follow up. Wound assessment over time has shown supple skin that has been resistant to trauma and infection. The cosmetic results were judged to be fair to good by surgeons and patients after one year's follow up.

Effect of Local Massage on Vaccination: DTP and DTPa

We previously demonstrated that local massage for one minute can enhance immunogenicity of diphtheria, tetanus, and whole-cell pertussis (DTPw) vaccination. This study further analyzes the effects of more intense local manipulation on infants after DTPw and DTPa (diphtheria, tetanus, and acellular pertussis) vaccination. A total of 808 infants aged two months were recruited to be vaccinated with either DTPw or DTPa. Vaccinees in both groups were further divided into two groups; those receiving local manipulation (massage and hot packing after vaccinations) and those receiving only vaccinations. Results showed that safety profiles were largely similar between those who had local manipulation following vaccination and those without. The only significant difference was more frequent local reactions including pain and swelling following the first two doses in both the DTPa and DTPw groups receiving manipulation compared with the groups not receiving manipulation. Serologic tests revealed that local manipulation had no significant effect on antibody response to pertussis toxin and filamentous hemagglutinin, and diphtheria and tetanus toxins. The effect of local massage on DTPw was related to the intensity of local massage. Too vigorous a local manipulation caused adverse local reactions and no beneficial effect on antibody response. As for the infants receiving DTPa and local massages for two minutes with hot packing, no significant effect on either the reactogenicity or immunogenicily was found.

Preliminary Study of Human Blood Substitute Looks Promising

Attempts to create human blood substitutes using dissociable tetrameric acellular hemoglobin have shown unacceptable rates of renal and cardiovascular

Production of DL-DOPA from acellular slime-mould

Production of DL-DOPA from acellular slime-mould

Suppression of spermatogenesis by low-level glycerol treatment.

Previous studies have shown that a single intratesticular injection with 70% glycerol results in rapid and long-term suppression of spermatogenesis. Because of the extensive testicular damage in the form of focal destruction and large numbers of acellular seminiferous tubules, it was difficult to examine the possible chemical mechanisms of antispermatogenic action. Our objective was to explore regimens of treatment that would result in significant suppression of spermatogenesis without significant testicular damage. Sexually mature Sprague-Dawley rats were injected intratesticularly with various glycerol concentrations (0, 10, 20, 40, or 70%) and with volumes of either 200 or 350 microliters per testis. At times between 1 and 36 weeks after the injection, the effect on weights of testes and accessory sex organs, testicular histology, sperm numbers, serum hormonal levels, and gonadotropin-receptor binding was examined. An injection of 350 microliters/testis of a 10% glycerol solution led to a significant decrease in weights of testes and epididymides. The treatment resulted in an overall suppression of spermatogenesis, with about 90% fewer sperm in the epididymides than in control animals. Histologically, the treatment decreased the number of normal tubules from 97% (control) to 16% and resulted in testes in which about 75% of the tubules were aspermatogenic (containing only Sertoli cells and spermatogonia). The number of acellular tubules (tubules without cytological detail) was generally less than 5% of the total, and there was negligible focal destruction. Serum levels of gonadotropins and androgens were not altered significantly by the treatment, and Sertoli cell glutamyl transpeptidase activity appeared normal. An equi-osmolar solution of glucose also resulted in significant suppression of spermatogenesis, but the effect of glycerol was significantly greater, suggesting a mechanism in addition to hyperosmolarity. This study, therefore, is further evidence for the specificity of glycerol actions and for its potential as an antispermatogenic agent.

Acellular and Whole-Cell Pertussis Vaccines as Booster Doses: A Multicenter Study

To compare the safety and immunogenicity of a variety of acellular (AC) and whole-cell (WC) pertussis vaccines combined with diphtheria and tetanus toxoids. Standard enrollment and reaction forms were used at five sites, and serologic evaluation was performed at a single site. Nine AC (Massachusetts Public Health Laboratories, Biocine Sclavo recombinant pertussis toxoid [PT], Connaught/BIKEN, Lederle three-component, Biocine Sclavo recombinant three-component, SmithKline Beecham three-component, Porton three-component, Takeda-Wyeth, and Connaught multicomponent), and three WC (Connaught Laboratories, Lederle Laboratories, and Massachusetts Public Health Laboratories) were studied. All AC contained varying concentrations of PT; some vaccines also contained filamentous hemagglutinin (FHA), pertactin, and/or agglutinogens. Two hundred forty children, aged 16 to 21 months and 4 to 6 years, were enrolled at five sites. Significantly less fever, redness, swelling, pain, limp, and use of pain medication were noted following AC compared with WC. Significant increases in antibody to PT were seen following all vaccines. Significant rises in FHA antibody were seen following all WC and the seven AC that contained FHA. Postbooster PT antibody levels were similar among the AC groups, regardless of the amount of PT administered (between 3.5 and 25 micrograms per dose). The dose of FHA did not affect PT antibody response. Infants primed with WC who were boosted with a monocomponent PT vaccine did not manifest a significant antibody response to FHA. The rate of adverse reactions was not a function of the number of antigens or the antigen quantity in the acellular vaccines, and antibody responses following AC were similar or better than antibody responses following WC. These results support the further evaluation of these vaccines in a larger National Institute of Allergy and Infectious Diseases-sponsored study in infants.

Three-dimensional architecture of the mesangial matrix--comparison of the intact and acellular glomerulus.

The three-dimensional ultrastructure of the rat mesangial matrix was studied in acellular rat renal cortex in comparison with intact cortex by scanning electron microscopy (SEM). The mesangial region was covered with fenestrated endothelial cells and was not identified in untreated specimens by SEM. Acellular renal cortex was obtained by perfusion with 4 mM EDTA, 3% Triton X-100, 0.0025% deoxyribonuclease in 1 M NaCl and 4% sodium deoxycholate. Transmission electron microscopy revealed that the glomerular basement membrane (GBM) and mesangial matrix maintained their respective shape and did not collapse even after removal of the cellular components. By SEM, the mesangial matrix appeared as fenestrated septa with oval or round stomata between the glomerular capillaries. The diameter of the stomata was 601 +/- 290 nm. The diameter of the fibrils forming the stomata was 177 +/- 80 nm. These fenestrated structures of the mesangial matrix appear to be useful in supporting glomerular tufts, in stretching and retracting mesangial cells and in passing macromolecules from the capillary lumen into the mesangium.

An in vitro model for polymorphonuclear-leukocyte-induced injury to an extracellular matrix. Relative contribution of oxidants and elastase to fibronectin release from amnionic membranes.

Alteration of the extracellular matrix by inflammatory cells is believed to be important in both lung injury and the subsequent restoration of lung architecture. Here we describe the results of the interaction between an acellular human amnionic membrane model and stimulated human polymorphonuclear neutrophils (PMN) in vitro. Polymorphonuclear neutrophil suspensions were placed on one surface of the amnion, and either the chemotactic peptide FMLP or the cell membrane activator phorbol myristate acetate (PMA) was placed on the opposite side of the amnion. Stroma and basement membrane sides of the amnion were separately exposed to the PMN. The PMN suspension was removed and centrifuged, and the supernatant was assayed for superoxide anion (O2-.) and for elastase activity. Injury to the acellular amnion was evaluated by transmission electron microscopy and by measurement of fibronectin (FN) released from the membrane matrix. Although both stimulants cause a concentration-dependent release of O2-., only PMA stimulated elastase release. These effects were similar when either the stroma or the basement membrane side was exposed to PMN. PMA-stimulated cells and supernatants from PMA-stimulated cells caused solubilization of membrane at different incubation times. Electron microscopy confirmed the disruption of the basement membrane of the amnion by PMA-stimulated PMN. Oxidant scavengers (SOD and catalase) did not prevent matrix degradation, and elastase inhibition by a specific chloromethylketone inhibitor diminished FN release on both sides of the amnion by activated PMN supernatants, but only on the basement membrane side by intact PMN. We conclude that in this model, elastase rather than oxygen radicals solubilizes FN from the matrix.

Retention of intact cells on bronchoscopy brushes after cleaning.

Reusable nylon bristle bronchoscopy brushes were evaluated to assess whether cytologically recognizable cells could be retained on the bristles after conventional cleaning techniques. Twenty brushes were used during bronchoscopy; 5 brushes were inserted directly into freshly resected neoplasms. The brushes were then scrubbed and rinsed in chlorhexidine gluconate, iodine, and alcohol, and air dried. Several days later fresh cytologic specimens were prepared from the apparently clean brushes. Eighty percent of the cytologic specimens exhibited strands of mucus, most containing well-preserved alveolar macrophages, polymorphonuclear leukocytes, and ciliated epithelial cells. Although no malignant cells were retained, it is clear that conventional cleaning techniques are inadequate for the removal of cells and acellular debris. Consideration should be given to immersing brushes in acetylcysteine or employing disposable brushes.

Native banded collagen fibrils in the glomerular mesangial matrix of normal human and laboratory animals

Renal cortical tissue blocks were removed from normal freshly harvested rat, rabbit, sheep, monkey, and human kidneys. The tissues were made acellular by extraction with distilled water and detergents. The resultant extracellular matrices and associated basement membranes (BMs) were observed by transmission electron microscopy. BMs and noncellular components of the interstitium maintained their in vivo histoarchitectural arrangements. Glomerular BMs were particularly striking in this regard and exhibited capillary loops radiating from centrolobular zones that contained a fibrillogranular mesangial matrix of medium electron density. An unexpected finding was the presence of bundles of banded collagen fibrils (45-nm repeat periods) embedded in the glomerular mesangial matrix in each of the species studied. These fibrils were morphologically indistinguishable from those seen in the general renal connective tissue space. Our data suggest that interstitial collagens may be present in substantial quantities in normal renal centrolobular zones. Furthermore, they may contribute to the unique intrinsic structural rigidity exhibited by isolated glomerular BMs.