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At least 19 recordsLinked to original sources
PubMed2025

Rapidly decellularized adipose tissue induces soft tissue vascularization in potential anatomical spaces.

Decellularized tissues provide biological cues owing to the wealth of structural and regulatory factors that promote angiogenesis, adipogenesis, and myogenesis and facilitate neurite outgrowth. Here, we demonstrated the advantages of decellularized adipose tissue (adipoECM) over defined collagen-based biomaterials for host tissue integration. Three batches of human adipose tissue were decellularized using a rapid decellularization protocol and analyzed using mass spectrometry. To assess the biological activity of the decellularized materials, adipoECM and a reference standard of care biomaterial (Integra®DRT, also containing collagen I and glycosaminoglycans) were implanted subcutaneously, but far from the wound bed (in anatomical potential spaces) of immunocompetent BALB/c mice. The mice were euthanized in the acute (1 day) and chronic (day 60) inflammatory reaction phases, followed by biomaterial excision and Masson’s trichrome immunohistofluorescence imaging of the paraffin-embedded specimens. Each batch of processed tissue passed a quality control check, showing a low level of donor genomic DNA, lack of nuclei, lipids, endotoxins, and bacterial contamination. Mass spectrometry revealed that all batches of decellularized tissue mainly contained collagen I and, to a lesser degree, collagen III, collagen IV, collagen V, laminin, fibrillin, fibronectin, tenascin, and elastin. No acute inflammatory reaction was observed in either material one day post-transplantation. At 60 days post-implantation, different cell types were detected in adipoECM specimens, whereas Integra®DRT remained acellular. Additional immunohistochemical staining of adipoECM revealed CD31-positive cells in the blood vessels. Mesenchymal (CD90 positive) and myeloid (CD14 positive) cells were also detected. Primary cell types involved in soft tissue healing and remodeling were found in the adipoECM-treated group. The ingrowth of blood vessels and mesenchymal cells confirmed the effective integration of adipoECM with host tissues. Our results demonstrate that decellularized adipose tissue implanted away from the wound bed possesses contextual biological activities that promote efficient integration with host tissues.

Adipose Tissue↗
PubMed1992

Epithelial and extracellular matrix injury in quartz-inflamed lung: role of the alveolar macrophage.

The bronchoalveolar leukocytes from quartz-inflamed lung were separated into macrophage-enriched and neutrophil-enriched populations on density gradients. Neutrophil-enriched populations showed the greatest activity in causing injury to epithelial cells and fibronectin in vitro. Inflammatory macrophage-enriched populations from quartz-exposed lung had the ability to cause fibronectin degradation but could not cause detachment injury to epithelial cells over and above that caused by control alveolar macrophages. Fibronectin damage in vivo could be an important factor in disordering the connective tissue scaffold of the lung, thereby favoring fibrosis. In vitro quartz stimulated more production of cytokines by alveolar macrophages than the inert particulate titanium dioxide. Cytokines could be important in upregulating adhesion molecules in the membranes of lung cells in vivo; this process could aid leukocyte/lung cell contact, allowing epithelial injury to be expressed, and could also be a factor leading to pathological change.

Animals↗
PubMed2016

A synergistic approach to the design, fabrication and evaluation of 3D printed micro and nano featured scaffolds for vascularized bone tissue repair.

3D bioprinting has begun to show great promise in advancing the development of functional tissue/organ replacements. However, to realize the true potential of 3D bioprinted tissues for clinical use requires the fabrication of an interconnected and effective vascular network. Solving this challenge is critical, as human tissue relies on an adequate network of blood vessels to transport oxygen, nutrients, other chemicals, biological factors and waste, in and out of the tissue. Here, we have successfully designed and printed a series of novel 3D bone scaffolds with both bone formation supporting structures and highly interconnected 3D microvascular mimicking channels, for efficient and enhanced osteogenic bone regeneration as well as vascular cell growth. Using a chemical functionalization process, we have conjugated our samples with nano hydroxyapatite (nHA), for the creation of novel micro and nano featured devices for vascularized bone growth. We evaluated our scaffolds with mechanical testing, hydrodynamic measurements and in vitro human mesenchymal stem cell (hMSC) adhesion (4 h), proliferation (1, 3 and 5 d) and osteogenic differentiation (1, 2 and 3 weeks). These tests confirmed bone-like physical properties and vascular-like flow profiles, as well as demonstrated enhanced hMSC adhesion, proliferation and osteogenic differentiation. Additional in vitro experiments with human umbilical vein endothelial cells also demonstrated improved vascular cell growth, migration and organization on micro-nano featured scaffolds.

Bone Regeneration↗
PubMed2026

Transforming Curcuma longa leaf waste into cellulose scaffolds.

The constant dearth of transplantable tissues and organs in India required the development of substitute biomaterials for tissue engineering. Plant-based decellularized scaffolds have become attractive options because of their abundance, ethical acceptability, architectural diversity, and lower risks of zoonotic transmission. Curcuma longa leaves were investigated in this study as a possible source of cellulose-based scaffolding for use in biomedical applications. After cuticle removal, an immersion decellularization technique utilizing sodium dodecyl sulphate (SDS) and triton-X-100 was developed to successfully remove cellular and nuclear material while maintaining leaf parenchyma architecture. Histology, DAPI staining, scanning electron microscopy, and a notable decrease in leftover DNA content all demonstrated efficient decellularization. When contrasted with native leaves, the resultant decellularized C. longa leaf scaffolds showed significant increase in porosity, water vapor transmission rate and swelling percent, and significantly lower contact angle with an optimum surface roughness promoting cell adhesion. Mechanical test manifest higher tensile strength with decreased stiffness. Fourier transform infrared spectra of leaf scaffold reveals persistence of different components except cuticle but the intensity of different peaks was decreased. The leaf scaffolds showed superior hemocompatibility and excellent compatibility with Madin-Darby canine kidney cells (MDCK) which is demonstrated by cell attachment and proliferation. MTT assay of seeded scaffold showed significantly higher metabolically active cell. In vivo subcutaneous implantation of decellularized scaffolds showed host tissue incorporation, accumulation of collagen, and neovascularization. C. longa leaf scaffolds can be utilized as cost effective and sustainable biomaterials for soft tissue engineering and regenerative medicine.

Curcuma↗
PubMed2026

Mesenchymal Stem Cell-Derived Exosomes Combined With 3-Dimensional Hyaluronan-Based Scaffold Promote Tendon-to-Bone Tunnel Healing.

PURPOSE: Tendon-to-bone healing remains a major clinical challenge due to poor regenerative capacity at the enthesis. This study aimed to evaluate the effects of mesenchymal stem cell-derived exosomes combined with a 3-dimensional hyaluronan-based scaffold on graft healing within bone tunnels. This study was conducted in accordance with the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines. METHODS: A total of 128 tendon-bone models were created in 64 Sprague-Dawley rats, randomized into four groups: control, exosome-only, scaffold-only, and exosome-loaded scaffold. At weeks 4 and 8 postoperatively, samples were analyzed histologically (hematoxylin-eosin, Masson Trichrome), immunohistochemically (fibroblast growth factor 2, bone morphogenetic protein 2), and biomechanically (maximum failure load). RESULTS: At both time points, the exosome-loaded scaffold group demonstrated significantly enhanced vascularization, cellular activity, and collagen fiber continuity and parallelism compared to all other groups (P < .05). Fibroblast growth factor 2 and bone morphogenetic protein 2 expression levels were highest in the exosome-loaded scaffold group, indicating early activation of proregenerative pathways. Biomechanically, this group also exhibited the greatest maximum failure load (15.64 &#xb1; 0.86 N at week 4; 22.97 &#xb1; 2.86 N at week 8), suggesting superior tendon-to-bone integration. The exosome-only group showed delayed but comparable improvements by week 8. CONCLUSIONS: Combining mesenchymal stem cell-derived exosomes with a 3-dimensional hyaluronan-based polycaprolactone/tricalcium phosphate scaffold enhances early and sustained healing at the tendon-bone interface. This cell-free, biocompatible strategy significantly improves vascularization, growth factor expression, collagen organization, and mechanical strength. These findings support its potential as a clinically translatable approach for improving tendon-to-bone healing outcomes. TYPE OF STUDY/LEVEL OF EVIDENCE: Therapeutic V.

Animals↗
PubMed1992

[An experimental study on tissue induction in anterior cruciate ligament reconstruction with the scaffold-type polyester artificial ligament (Leeds-Keio)].

The effect of experimental autogenous tissue implantation (infrapatellar fat pad, fascia lata) together with scaffold type artificial ligament (Leeds-Keio) in ACL reconstruction was studied in 56 mongrel adult dogs. Specimens were examined macroscopically, histologically and by means of microangiography. The results were compared with those in which the artificial ligament was used alone. When infrapatellar fat pad was transferred on artificial ligament, tissue induction was observed 2 weeks after reconstruction. When ACL was reconstructed using a combination of artificial ligament and a small piece of fascia lata, maturated dense collagen fibers were observed 24 weeks after reconstruction. These results indicated that quicker tissue induction could be obtained in ACL reconstruction using an artificial ligament together with a infrapatellar fat pad, and that collagen fiber maturation was accelerated when ACL was reconstructed using an artificial ligament together with a small piece of fascia lata.

Adipose Tissue↗
PubMed1975

Evidence for successful acceptance of irradiated free gingival allografts in dogs.

Free graft samples were excised via split thickness dissection from two mongrel dogs, washed in lactated Ringer's solution, immersed into tubes containing glycerol and distilled water, and frozen to -55 degrees C. Subsequently the grafts were exposed to 2.5 x 10-6 rads of 60-Co gamma-radiation. The irradiated allogeneic grafts were later reconstituted and surgically transferred to four recipient subjects. Three autogenous nonirradiated grafts were also placed as controls. The animals were killed so as to furnish healing data at 0, 3, 7, 10, and 40 days postoperatively. Fourteen allografts were evaluated. They were judged to be nonantigenic, immunologically incompetent and nonviable. However, retention of an essentially unaltered connective tissue corium may have been instrumental in supporting subsequent epithelial regeneration from adjacent host tissue, while being passively incorporated into a very dynamic receptor zone. The graft thus served as a scaffolding for connective tissue deposition and attachment. It appears that the experimental regime obviated the immunologic interference usually encountered in allografting procedures. Thus, high intensity irradiation of the graft tissue may have rendered the tissue to be immunologically tolerable. Further studies are required to ascertain the duration of the host's immunologic unresponsiveness to the alien tissue, as well as ultimate structural and biologic fate of the transplanted tissues. Experiments are now in progress which have been designed to test the extent of immunologic sensitization induced by the grafted tissue.

Animals↗
PubMed1992

[Reconstruction for malignant bone tumor defects by a composite graft--cementless arthroplasty].

Five patients with malignant bone tumor were arthroplastied with Dacron fabric-enveloped prosthesis without using cement after resection of tumor. Usually following the wide resection of the primary lesion, the supporting tissues are always lost. Dacron fabric is intended to act as a scaffold upon which connective tissue can proliferate and form new ligament and supporting soft tissues. Dacron fabric is useful for the repair of ligament, muscles, joint capsule, periosteum, and other supporting soft tissues without significant complications. The lengths of the released supporting tissues are easily adjustable. Biologic fixation of the prosthesis to the soft supporting tissues is successful, then the durability of the prosthesis prolongs. The Dacron fabric is capable of inducing and supporting the ingrowth of vascular connective tissue which becomes stronger thereafter. The author believes that this method provides a stable, functional and esthetically pleasing reconstruction.

Adult↗
PubMed2026

Single-Cell Triomics Analysis of Tumor Cells Infiltrating Patient-Derived Breast Cancer Scaffolds.

Cellular heterogeneity plays a critical role in tissues and diseases, including cancer. Single-cell technologies are required to provide detailed information about the phenotype and genotype of individual cells. Despite several approaches to analyzing different analytes at the single-cell level, it is challenging to assess DNA, RNA, and protein simultaneously. Here, a single-cell triomics method to assess DNA, RNA, and proteins from the same cell using a targeted sequencing approach is shown. Breast cancer cells cultured in monolayers and in patient-derived scaffolds that mimic in vivo-like growth conditions, both with and without chemotherapy treatment, were analyzed. Data showed that DNA, RNA, and protein biomarkers could be reliably analyzed, providing biological insights into breast cancer cell heterogeneity. In addition, chemotherapy treatment caused changes in subpopulations and expressions of biomarkers. Furthermore, cells growing in patient-derived scaffolds generated from various breast cancers affected cell heterogeneity and drug resistance differently as a result of the unique tumor-specific microenvironments. The data show that single-cell triomics provides new means to assess cancer cell heterogeneity at DNA, RNA, and protein levels.

Humans↗
PubMed1975

Bone remodelling in rabbit ear chambers. A vital microscopical and histological study.

Heterotopic autologous bone tissue was studied by vital microscopy in rabbit ear chambers and correlated with the findings from histological sections prepared from the same tissue. The in vivo morphology and remodelling of different bone types were thereby characterized. Autografts were resorbed in a disordered fashion by osteoclasts and woven-fibred bone tissue formed on such grafts. Later parallel-fibred bone, bounded by a seam of osteoid tissue, formed on this scaffold. Bone flakes growing underneath the cover glass were remodelled into shallow hemi-ossicles containing fat marrow, which were always oriented towards the cover glass. Following en bloc removal of the central part of the bone culture, woven-fibred bone rapidly formed within the new connective tissue, filling in the empty space. Such bone was remodelled into cancellous bone containing fat marrow. A balance between resorption and formation was regularly established in the chambers. The bone cultures were thus useful for vital microscopical studies for half a year or longer. In the present study bone remodelling in rabbit ear chambers closely resembled that of orthotopics bone. Ear chamber bone cultures are therefore a versatile in vivo biological model for dynamic bone tissue studies at both tissue and cellular levels.

Animals↗
PubMed2026

FBN1-related connective tissue disorders: unraveling cardiovascular, skeletal, and ocular complications through TGF-&#x3b2; signaling dysregulation and genotypic correlations.

Fibrillin-1 is an extracellular matrix glycoprotein essential for microfibril integrity, mediating cell-matrix interactions, providing structural support to tissues, and serving as a scaffold for elastogenesis. Pathogenic variants in the fibrillin 1 gene (FBN1) give rise to a spectrum of autosomal dominant connective tissue disorders collectively termed type-1 fibrillinopathies, which include Marfan syndrome, geleophysic dysplasia 2, acromicric dysplasia, Weill-Marchesani syndrome 2, marfanoid-progeroid-lipodystrophy syndrome, stiff skin syndrome, MASS syndrome, and isolated ectopia lentis 1. These disorders predominantly manifest cardiovascular, skeletal, and ocular abnormalities. Among these, aortic and valvular lesions are the principal and most life-threatening complications and therefore warrant the greatest clinical attention. Skeletal anomalies are diverse and can even be diametrically opposed across different phenotypes, while ectopia lentis represents the hallmark of ocular conditions. Notably, mutant fibrillin-1 disrupts microfibril structure and/or function, leading to dysregulated transforming growth factor-&#x3b2; (TGF-&#x3b2;) signaling, which is widely recognized as a central mechanism underlying type-1 fibrillinopathies. Although numerous pathogenic FBN1 variants have been identified, the knowledge of genotype-phenotype correlations remains limited in some specific regions. This review synthesizes the current understanding of the FBN1-related molecular mechanisms linking aberrant TGF-&#x3b2; signaling to distinct phenotypic outcomes and discusses how genetically engineered animal models and human induced pluripotent stem cell models advance mechanistic insights and facilitate therapy development. Additionally, clinical manifestations and genetic characteristics across all phenotypes are elaborated to facilitate diagnosis, treatment, and management of these complex disorders.

Cardiovascular complications↗
PubMed1992

Adenocarcinoma-reactive human monoclonal antibody MS2B6 defines an antigen in simple glandular epithelium.

A human monoclonal antibody (MAb), MS2B6, produced from splenocytes isolated from a patient with advanced papillary serous cystadenocarcinoma of the ovary, defines a unique human tumor-associated antigen. This antigen, EA2B6 (epithelial antigen 2B6), is expressed in a tissue-restricted manner on cultured and fresh human adenocarcinomas and some normal glandular epithelial tissues. EA2B6 is a 38-48 kD protein antigen that co-fractionates with the nuclear matrix-intermediate filament scaffold of simple glandular epithelial tissues. EA2B6 is a molecule with restricted solubility, and in vitro antigen-antibody binding is dependent on the antigen being presented on a solid support. To determine if EA2B6 is a cytokeratin, competition studies were undertaken with several cytokeratin-specific murine monoclonal antibodies. None of these antibodies inhibited the binding of human MAb MS2B6 to partially purified EA2B6. Less than 1% of HT29 colon adenocarcinoma cells and fresh ovarian adenocarcinoma ascites cells express EA2B6 on their surface. The majority of EA2B6 is intracellular. Because of the restricted tissue distribution of this antigen and stability of the antibody, we believe MS2B6 is a good candidate for MAb-mediated diagnosis and therapy of human adenocarcinomas.

Adenocarcinoma↗
PubMed1992

Regeneration of pericardial tissue on absorbable polymer patches implanted into the pericardial sac. An immunohistochemical, ultrastructural and biochemical study in the sheep.

A new absorbable polymer prepared from polyhydroxybutyrate (PHB) was inserted as a pericardial patch in sheep to serve as a temporary scaffold for regeneration of pericardial tissue. Postoperative adhesions were rare or absent. The present study focuses on characterization of the regenerated surface cells. The luminal surface of the regenerated tissue was covered with a complete layer of mesothelium-like cells which at light and scanning electron microscopy resembled those in native pericardium. Immunohistochemical stainings for cytokeratin and thrombomodulin were positive in these cells. Heparan sulfate proteoglycan was found in a basement-membrane-like structure beneath the surface cells, as in the normal pericardium. Transmission electron microscopy of the regenerated surface revealed cells with the characteristics of mesothelium. Prostacyclin production in the regenerated tissue was similar to that in native pericardium. The results indicate regeneration of a mesothelial layer with many of the important functions of native mesothelial cells. This may explain the presently and previously observed prevention of pericardial adhesions after cardiac surgery in this field. Clinical testing of PHB patches as pericardial substitutes is warranted in cardiac surgery when pericardial closure is desired.

Animals↗
PubMed2026

Cucurbitacins in Plant-Insect Interactions: Biosynthesis, Regulation, Ecological Functions, and Prospects for Crop Protection.

Cucurbitacins are highly oxygenated tetracyclic triterpenoids characterized by intense bitterness, substantial structural diversity, and important consequences for plant-herbivore interactions. Although best known from Cucurbitaceae, cucurbitacins and related cucurbitane-type metabolites also occur in phylogenetically distant herbaceous and woody plants. Genetic and biochemical studies have validated several core biosynthetic steps, including cucurbitadienol formation by oxidosqualene cyclases and subsequent modification by cytochrome P450 monooxygenases, acyltransferases, and glycosyltransferases. Tissue-preferential basic helix-loop-helix transcription factors constitute the best-characterized regulatory layer, whereas the evidence supporting accessory regulators, transporters, and environmental responses varies from functional validation to transcriptomic or genomic prediction. From the plant perspective, cucurbitacins deter feeding or impair performance in many generalist and non-adapted herbivores. By contrast, their use as host-recognition cues and feeding stimulants by specialist diabroticite beetles reflects evolved herbivore adaptations involving perception, tolerance, metabolism, or sequestration rather than a second defensive function of the plant trait. Herbivore-induced cucurbitacin accumulation has been demonstrated in particular systems, although its regulatory mechanisms and ecological generality remain unresolved. Unlike previous reviews centered primarily on cucurbitacin chemistry, pharmacological activity, or individual biosynthetic pathways, this review integrates evidence-graded pathway reconstruction and molecular regulation with taxonomic distribution, insect adaptation, domestication, and agroecological consequences. Mechanistically, this review traces how scaffold formation, oxidative tailoring, conjugation, tissue-specific regulation, and transport give rise to contrasting ecological outcomes through herbivore-specific perception, tolerance, metabolism, and sequestration. We conclude that uniformly increasing or eliminating cucurbitacins is unlikely to provide broadly effective crop resistance because either direction may favor a different herbivore group. Future priorities include functional validation of candidate genes, spatially resolved metabolite analysis, comparative investigation of non-cucurbit lineages, and field evaluation involving generalist and specialist herbivores, crop quality, and non-target organisms. These advances will support context-specific fruit-quality improvement, behavioral pest control, and integrated pest management strategies rather than cucurbitacin manipulation as a stand-alone resistance approach.

agroecology↗
PubMed1979

Fibronectin distribution in epithelial and associated tissues of the rat.

Specific antiserum was used to investigate the distribution of the extracellular glycoprotein, fibronectin, in rat skin and tongue tissue by light and electron microscopy with immunofluorescence and immunoperoxidase techniques. We conclude that fibronectin is absent from stable, differentiated parts of tissues, such as the sebaceous glands or the matrix, medulla, cortex, and cuticles of the hair and the inner and outer root sheaths, or even in tissues in which there is some cell movement, such as the epidermis. It is, however, characteristic of sites at which cell division is occurring in contact with an extracellular scaffolding, such as basement membrane or loose connective tissue. Conspicuous examples were in the glassy membrane and connective tissue sheath associated with the follicular epithelium, the basement membrane underlying vascular endothelial cells, the connective tissues surrounding and investing nerve and muscle fibre bundles, and the dermal connective tissue where fibronectin was often associated closely with collagen fibres. At the basement membrane of the dermal/epidermal junction, fibronectin occurred at the plasma membrane of the basal cells and in the lamina lucida area. There was no correlation with specific areas of cell-substrate adhesion, such as the hemidesmosomes. The endoplasmic reticulum of fibroblasts stained strongly suggesting that these cells represent a major site of synthesis.

Animals↗
PubMed2026

Hex-MASP for mapping the whole-tissue spatial proteome and the intrabrain distribution of monoclonal antibodies.

Whole-tissue level spatial proteomics provides critical insights into region-specific biological regulations but remains challenging. Previously, we introduced the micro-scaffold assisted spatial proteomics (MASP) concept for whole-tissue mapping. However, this prototype required substantial development in spatial resolution, practicality, and throughput for practical application. Here we present a next-generation MASP technique (hex-MASP) featuring i) a new design of hexagonal-micro-wells fabricated with optimized projection micro-stereolithography 3D-printing, achieving high spatial resolution, sampling robustness, and mechanical strength for reproducibly compartmentalizing even tough tissues; ii) enhanced throughput/effectiveness in sample preparation and LC-MS analysis with high quantitative quality. Applied to mouse brain, hex-MASP achieved in-depth, whole-tissue mapping for >6,000 proteins in mouse brains, with high spatial accuracy and excellent data quality. The substantially improved resolution revealed critical regional details across the entire brain, that were not previously captured, enabling precise depiction of protein distribution heterogeneity. This technique enabled the identification of many unreported regionally enriched proteins across brain structures. We further applied hex-MASP to investigate the intrabrain distribution of intracerebroventricularly dosed antibody therapeutics and related proteins, which enabled whole-tissue mapping of protein drugs revealed insights into antibody brain penetration and distribution. Hex-MASP represents a robust, scalable platform for whole-tissue spatial proteomics.

Animals↗
PubMed1992

Morphometry of cupromeronic blue-stained proteoglycan molecules in animal corneas, versus that of purified proteoglycans stained in vitro, implies that tertiary structures contribute to corneal ultrastructure.

Isolated, purified small chondroitin (dermatan) sulphate proteoglycans from corneas of cow and rabbit and cow sclera were stained with Cupromeronic blue in 'model' experiments. The lengths and thicknesses of the images were compared with those of the same proteoglycans stained in the tissue, using the critical electrolyte concentration principle to give specificity for sulphated proteoglycans, and keratanase 1 or chondroitinase ABC digestion to distinguish between chondroitin and keratan sulphate. Corrections for orientation of the stained glycan filaments within the section plane were made to convert the observed lengths to true average lengths. Observed lengths of stained chondroitin (dermatan) sulphate were greater than those of keratan sulphate, both in models and tissues, in agreement with published data from biochemical and rotary-shadowing studies, in both species. Corrected (true) average lengths of stained isolated chondroitin (dermatan) sulphate proteoglycans were slightly, but not significantly, longer than expected from rotary shadowing or biochemical measurements. Keratan sulphate lengths were similarly somewhat longer. The data support the idea that Cupromeronic blue acts as a scaffold that helps maintain polyanion shape against distortion on staining. Stained filaments in tissues were sometimes over twice the length of isolated stained proteoglycans, suggesting that 2 glycan chains were aligned end-to-end. Thicknesses of proteoglycan filaments suggested that at least 2 glycan chains were aligned side-by-side, both in models and in tissues. A scheme for proteoglycan tertiary structure in cornea is proposed, in which glycan chains may bridge collagen fibrils in duplexed forms similar to those observed in rotary shadowed preparations.

Animals↗