Bone healing after amputation of mouse digits and newt limbs: implications for induced regeneration in mammals

Postamputational healing was compared in nonregenerating and regenerating animals to determine whether bone healing might interfere with a regenerative response in mice. More than 150 mouse toes and 100 newt limbs were examined at the light microscope level. Stages of normal bone healing with approximate times of occurrence were established. Major differences in healing of these two species were seen. The periosteum produced hyaline cartilage, woven bone, and chondroid bone in mice, but only hyaline cartilage in newts. The endosteum produced woven bone in mice but no new growth in newts. Dead bone persisted in mice but was removed in newts. The marrow cavity became sealed in mice but remained open in newts. Despite these differences both animals produced skeletal tissue distal to the amputation plane. Woven bone formed distal to the amputation plane of mice. Cartilage formed distal to the amputation plane of newts, but cartilage was never seen distal to the plane of mice. Results of previous studies reveal that cartilage can be formed distal to the amputation plane of experimentally treated mice. Thus, although it does not regenerate, mouse bone is capable of producing, distal to the amputation plane, the type of skeletal tissue which appears at that location during an epimorphic regenerative response. This observation, in combination with other experimental results, indicates that both skeletal and soft tissues at the amputation site of treated mammals can resemble comparable tissues of newt limbs at an early stage of regeneration.     

Open finger tip healing and replacement after distal amputation in Rhesus monkey with comparison to limb regeneration in lower vertebrates

Summary The left thumbs and great toes of three 81/2 month old Rhesus monkeys (Macaca mulatta) were amputated in guillotine fashion one millimeter distal to the base of the nail and allowed to heal by the conservative open wound method. Healing occurred in seven to ten days in these small digits. Each of the thumbs and toes grew back with some blunting and shortening of the digit tips, but were functional. The new structures were cosmetically pleasing as in the human instances. The nails grew essentially to normal size and shape supported by the remaining portions of the distal phalanges. Histological studies showed no evidence of blastema formation such as is observed in the regenerating limb of the Urodele (newt) taken as the comparative representative. The possibility of improving the regrowth is discussed against the background of our knowledge of the importance of nerve during limb regeneration in lower vertebrates. Offprint requets to: M. Singer     

A cellular,molecular, and pharmacological basis for appendage regeneration in mice

Regenerative medicine aims to restore normal tissue architecture and function. However, the basis of tissue regeneration in mammalian solid organs remains undefined. Remarkably, mice lacking p21 fully regenerate injured ears without discernable scarring. Here we show that, in wild-type mice following tissue injury, stromal-derived factor-1 (Sdf1) is up-regulated in the wound epidermis and recruits Cxcr4-expressing leukocytes to the injury site. In p21-deficient mice, Sdf1 up-regulation and the subsequent recruitment of Cxcr4-expressing leukocytes are significantly diminished, thereby permitting scarless appendage regeneration. Lineage tracing demonstrates that this regeneration derives from fate-restricted progenitor cells. Pharmacological or genetic disruption of Sdf1–Cxcr4 signaling enhances tissue repair, including full reconstitution of tissue architecture and all cell types. Our findings identify signaling and cellular mechanisms underlying appendage regeneration in mice and suggest new therapeutic approaches for regenerative medicine.     

Structure,composition, and assembly of basement membrane

Basement membranes are thin layers of matrix separating parenchymal cells from connective tissue. Their ultrastructure consists of a three-dimensional network of irregular, fuzzy strands referred to as “cords”; the cord thickness averages 3–4 nm. Immunostaining reveals that the cords are composed of at least five substances: collagen IV, laminin, heparan sulfate proteoglycan, entactin, and fibronectin. Collagen IV has been identified as a filament of variable thickness persisting after the other components have been removed by plas-min digestion or salt extraction. Heparan sulfate proteoglycan appears as sets of two parallel lines, referred to as “double tracks,” which run at the surface of the cords. Laminin is detected in the cords as diffuse material within which thin wavy lines may be distinguished. The entactin and fibronectin present within the cords have not been identified as visible structures. The ability of laminin, heparan sulfate proteoglycan, fibronectin, and entactin to bind to collagen IV has been demonstrated by visualization with rotary shadowing and/or biochemical studies. Incubation of three of these substances–collagen IV, laminin (with small entactin contamination), and proteoglycan–at 35°C for 1 hr resulted in a precipitate that was sectioned for electron microscopic examination and processed for gold im-munolabeling for each of the three incubated substances. Three structures are present in the precipitate: (1) a lacework, exclusively composed of heparan sulfate proteoglycan in the form of two parallel lines, similar to double tracks; (2) semi-solid, irregular accumulations, composed of the three initial substances distributed on a cord network; and (3) convoluted sheets, which are also composed of the three initial substances distributed on a cord network but which, in addition, have the uniform appearance and thickness of the lamina densa of basement membrane. Hence these sheets are closely similar to the main component of authentic basement membranes.     

The isolation of cross-reactive monoclonal antibodies: hybridomas to streptococcal antigens cross-reactive with mammalian basement membrane

Based upon the assumption that post-streptococcal sequelae are the result of cross-reactive antibodies, hybridomas were prepared from the spleens of mice immunized with Group A type 12 streptococcal cell membranes (SCM) specifically to screen for such cross-reactive antibodies. One fusion produced a cell population displaying antibodies reactive to both SCM and glomerular basement membrane (GBM) antigens as demonstrated by ELISA technique. Ascites produced by this cell population also showed reactivity to lung basement membrane (LBM). Limiting dilution procedures have produced 15 monoclonal hybrids with both anti-SCM and anti-GBM activity. Confirmation of the cross-reactive and monoclonal nature of the antibody was accomplished by both direct and indirect competitive ELISA. These observations have established that unique cross-reactive antibody-secreting hybrid cells with reactivity to both SCM and basement membrane (BM) antigens can be isolated by standard cloning procedures.     

Comparison of non-collagenous type IV collagen subunits in human glomerular basement membrane, alveolar basement membrane, and placenta

This study examines the similarities and differences in the noncollagenous domain (NC1) of type IV collagen from human glomerular basement membrane (hGBM), alveolar basement membrane (hABM), and placenta (hPBM). Following collagenase digestion, NC1 domain was isolated on Bio-Gel A-0.5m or by cation exchange chromatography on S-Sepharose. NC1 from each source was characterized by SDS PAGE, and two dimension NEPHGE/SDS PAGE. Immunoblotting and ELISA inhibition was performed using antibody probes specific for M28 , M28+, M26 and M24 monomer subunits of human NC1. It was observed that all NC1 subunits were present in hGBM and hABM derived material, however M28 and M28+ monomers were absent in hPBM NC1. These findings indicate that while alpha 1(IV) and alpha 2(IV) collagen chains are present in hGBM, hABM and hPBM, alpha 3(IV) and alpha 4(IV) collagen chains are only found in hGBM and hABM but are absent in hPBM. It can now be appreciated that heterogeneity of alpha (IV) chain composition exists in basement membranes from various organs.     

Commentary: engineering of tissue healing and regeneration

Regenerative medicine aims to restore homeostasis of diseased tissues and organs. With time, engineered replacement tissue constructs will play an increasingly important role in achieving this goal. Equally important, however, will be the ability to resolve disease-associated inflammation and to optimize tissue regenerative capacity by specifically patterning the host tissue microenvironment. The tools of bioengineering are uniquely suited to meet these challenges. Here, the candidate molecular and cellular targets for manipulating the host's inflammatory environment and tissue regenerative capacity are briefly discussed within the context of current and emerging bioengineering strategies. The objective is to draw the attention of basic scientists and engineers to the importance of regulating inflammation in achieving the goals of tissue engineering and regenerative medicine.     

Age related carbohydrate content of mouse kidney glomerular basement membrane and its reactivity to antistreptococcal membrane antisera

Rabbit antisera to group A, type 12 streptococcal cell membrane (SCM) and human glomerular or glomerular basement membrane (GBM) preparations were found to immunologically react with adult mouse GBM by an indirect fluorescent antibody test. Such cross-reactivity was enhanced by enzymatic cleavage of GBM carbohydrate. In comparative tests with neonatal and adult kidneys, the antisera were more reactive with the former than the latter. GBM carbohydrate cleavage had little or no effect on the reactivity of neonatal GBM with the antisera. Quantitative carbohydrate analyses revealed that adult mouse GBM contained a greater content of carbohydrate than did neonatal GBM. Thus the differential reactivities of adult and neonatal mouse GBM with anti-SCM antisera are directly related to their respective carbohydrate contents.     

功能性组织工程皮肤附属物再生对排汗和新陈代谢的意义

背景：排汗可将一些溶解于汗液的代谢产物带出体外,在排泄中起辅助作用。现代医学目前还无法恢复严重创伤或烧伤患者的汗腺,如何解决皮肤汗腺的再生及如何培养具有皮肤附属物的“功能性组织工程皮肤”问题已成为当今医学研究中的热点。 目的：探讨机体的新陈代谢、汗液的代谢方式,分析组织工程皮肤功能性附属物汗腺再生研究的现状。 方法：收集组织工程皮肤功能性附属物再生,特别是排汗的相关研究,分析机体的新陈代谢与排泄方式,汗液代谢的物质和方式,以及目前组织工程皮肤附属物汗腺再生研究的现状。 结果与结论：汗液作为排泄系统中重要的一员在维持机体内部的动态平衡,防止代谢产物对身体的损害方面具有不可忽视的地位。理想的功能性组织工程皮肤应具有分泌汗液、排泄废物、调节体温和维持皮肤动态平衡的作用,在大面积烧伤后皮肤创伤修复和重建过程中起着至关重要的作用。已有研究将干细胞与汗腺细胞进行共培养,然后移植到瘢痕创面上进行重建汗腺组织,取得了一定的成功。     

Matrix metalloproteinase dysregulation in the stria vascularis of mice with Alport syndrome: implications for capillary basement membrane pathology

Alport syndrome results from mutations in genes encoding collagen alpha3(IV), alpha4(IV), or alpha5(IV) and is characterized by progressive glomerular disease associated with a high-frequency sensorineural hearing loss. Earlier studies of a gene knockout mouse model for Alport syndrome noted thickening of strial capillary basement membranes in the cochlea, suggesting that the stria vascularis is the primary site of cochlear pathogenesis. Here we combine a novel cochlear microdissection technique with molecular analyses to illustrate significant quantitative alterations in strial expression of mRNAs encoding matrix metalloproteinases-2, -9, -12, and -14. Gelatin zymography of extracts from the stria vascularis confirmed these findings. Treatment of Alport mice with a small molecule inhibitor of these matrix metalloproteinases exacerbated strial capillary basement membrane thickening, demonstrating that alterations in basement membrane metabolism result in matrix accumulation in the strial capillary basement membranes. This is the first demonstration of true quantitative analysis of specific mRNAs for matrix metalloproteinases in a cochlear microcompartment. Further, these data suggest that the altered basement membrane composition in Alport stria influences the expression of genes involved in basement membrane metabolism.     

Antigenic and morphologic regeneration of epidermal basement membrane in short term organ culture of human skin.

In fetal life the development of bullous pemphigoid antigen is preceded by a morphologically detectable basement membrane zone (BMZ). The present study investigated the possibility that similar morphologic and antigenic changes of the BMZ occur in short term organ culture of human skin. Twenty small pieces from each of four neonatal foreskin specimens were cultured under standard conditions. At regular intervals two pieces from each specimen were removed. One piece was frozen and cryostat sections were processed for indirect immunofluorescence using a patient's serum that was proven to have circulating pemphigoid IgG antibodies. Additional sections were stained with periodic acid-Schiff and hematoxylin and eosin. The other piece was processed for electron microscopic examination. Morphologically the BMZ started degenerating after 24 hours. Regeneration started on the 4th day and reached an optimal level on the 5th day. Chance of the immunofluorescence line at the BMZ began with a slight weakening at 24 hours followed by progressive widening and breakage until the 4th day when fluorescence was no longer detectable. Except for a few punctate areas immunofluorescence remained absent on the 5th day. Sixth day specimens, however, revealed reappearance of the immunofluorescence line in a continuous pattern. By the 7th day a second morphologic and antigenic degeneration started at the BMZ. This lag between the morphologic and antigenic regeneration of the BMZ in organ culture although shorter is comparable to the one observed during the development of the BMZ in fetal life.     

Involution and regeneration of the mouse thymus after lipopolysaccharide injection

N. I. Pirogov Second Moscow Medical Institute. (Presented by Academician of the Academy of Medical Sciences of the USSR V. V. Kupriyanov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 109, No. 3, pp. 280–283, March, 1990.     

Anti-glomerular basement membrane (GBM) glomerulonephritis in the mouse: development of disease and cell proliferation.

In a mouse model of anti-glomerular basement membrane (GBM) glomerulonephritis, associated with the nephrotic syndrome, a wide range of morphological and proliferative responses was seen in the renal corpuscle, at 6 days. The severity of the damage could be assessed by measuring the average daily weight gain between days 0 and 3 (DWG) of the animal. Those animals with a high DWG showed capsular proliferation, whereas animals with a low DWG showed predominantly tuft cell proliferation. Capsular cell birth rate increased with DWG whilst tuft cell birth rate was negatively related. A computer simulation suggests that the results are compatible with the induction of successive but overlapping waves of tuft and capsular cell proliferation.     

Regeneration of mouse lip epidermis after cryo treatment. Hemidesmosome formation and HSPG (heparan sulfate proteoglycan) distribution in basement membrane

The processes of degeneration and the regeneration of the lip epidermal cells was observed by electron microscopy, focussing on the substance and the structure of the lamina lucida, on which regenerating cells migrated. After the repetitive freezing and thawing treatment, epidermal cells degenerated and detached from the dermis. The separation occurred between the epidermal cells and the basement membrane, leaving a small amount of cell debris on the lamina densa. After the separation of the epidermis, there were some thick parts in the lamina densa which appeared to be the part below hemidesmosomes. Regenerating epidermal cells migrated from the nondegenerated area along the cellular surface of the old lamina densa. They migrated over the cell debris which was gradually phagocitized, and formed new hemidesmosomes with the old lamina densa. Regenerating epidermal cells did not make close contact with the old lamina densa during their migration, but there was a clear space in between, indicating that some of the materials and the structure of the lamina lucida of the old basement membrane was preserved. By immunoelectron microscopy using anti-HSPG (heparan sulfate proteoglycan) antibody, it became clear that after the epidermal separation, HSPG was preserved in the basement membrane to some extent, especially in the thick parts of the lamina densa located below. The immunoelectron micrographs support the view that hemidesmosomes may reform at the previous locations at the old lamina densa.     

Micropapillomatosis of the bronchial basement membrane: pathogenesis and types

Fingerlike excrescences of the bronchial basement membrane may occur as a consequence of alterations in human bronchial mucosa. Such lesions contain capillary vessels and subepithelial connective tissue in their interior (capillar body phenomenon, micropapillomatosis). The papillae develop passively as a result of capillary vessels pushing towards the epithelium. The precondition for the formation of papillae is an alteration of the epithelial layer from pseudostratified to stratified epithelium. The broad lower layer of the basement membrane is thinned due to the piling-up of wide-lumened capillary vessels at the basement membrane. The capillary vessel increases in length and forms loops which push the basement membrane up into the epithelial layer. In this process the epithelial cells are pushed apart. Four different types of papillae can be distinguished, the forms of which may be correlated with particular disturbances in the texture of the epithelial layer. Papillomatosis of the bronchial basement membrane is not pathological as such. It can contribute to the diagnosis of bronchus biopsy material. In the case of scaled-off epithelium its presence is evidence for an alteration of the mucosa into stratified epithelium (basal cell hyperplasia, squamous metaplasia, textureless epithelial layer).     

Collagenous and basement membrane proteins of chordoma: immunohistochemical analysis

Tissue localization of collagenous and basement membrane proteins in the extracellular matrix of five sacro-coccygeal chordomas and human fetal notochords was examined immunohistochemically to assess the implications for the histogenesis and histological diagnosis of chordoma. Human fetal notochords and conventional chordomas both exhibited basement membrane proteins (such as type IV collagen and laminin) and type VI collagen on the surfaces of cellular cords. Type II collagen, a main structural protein of cartilage, was also present in both tissues. In the chordomas, however, type II collagen was not so widespread as it was in the notochords, and the predominant collagenous protein was type I. In contrast, an altered deposition of these proteins was noticed in a recurrent tumour which, histologically, showed considerable atypia and eventually metastasized to the liver. The characteristic cartilage-type and basement membrane proteins disappeared and unusual collagen types, such as types III and V, appeared in the stroma. The results further support the notochordal origin of chordoma and suggest that the immunohistochemistry of collagenous and basement membrane proteins may be a helpful criterion for the histological diagnosis and prediction of the biological aggressiveness of chordomas.     

The mouse Mid1 gene: implications for the pathogenesis of Opitz syndrome and the evolution of the mammalian pseudoautosomal region

We have recently reported isolation of the gene responsible for X- linked Opitz G/BBB syndrome, a defect of midline development. MID1 is located on the distal short arm of the human X chromosome (Xp22. 3) and encodes a novel member of the B box family of zinc finger proteins. We have now cloned the murine homolog of MID1 and performed preliminary expression studies during development. Mid1 expression in undifferentiated cells in the central nervous, gastrointestinal and urogenital systems suggests that abnormal cell proliferation may underlie the defect in midline development characteristic of Opitz syndrome. We have also found that Mid1 is located within the mouse pseudoautosomal region (PAR) in Mus musculus , while it seems to be X- specific in Mus spretus. Therefore, Mid1 is likely to be a recent acquisition of the M. musculus PAR. Genetic and FISH analyses also demonstrated a high frequency of unequal crossovers in the murine PAR, creating spontaneous deletion/duplication events involving Mid1. These data provide evidence for the first time that genetic instability of the PAR may affect functionally important genes. In addition, we show that MID1 is the first example of a gene subject to X-inactivation in man while escaping it in mouse. These data contribute to a better understanding of the molecular content and evolution of the rodent PAR.