Effects of intersyringe processing on adipose tissue and its cellular components: implications in autologous fat grafting

Autologous fat grafting is a popular technique in plastic surgery. A mechanical processing method is used to facilitate fat injection. No study has investigated whether this process affects cell quality and preservation of biological functionality. This study analyzed the influence of quick mechanical processing through two interconnected small-diameter syringes ("shuffling") on both structure and viability of fat tissue, and on viability, clonogenicity, and differentiation of the freshly isolated stromal vascular fraction.; Lipoaspiration was performed in six healthy donors, followed by shuffling the fat either zero, five, or 30 times between two 10-cc syringes. Thereafter, fat was applied through a 1.5-mm cannula as in a clinical setting for autologous fat grafting. Analysis of different treatment conditions was conducted. Immunofluorescent staining allowed assessment of morphology, viability, composition, and damage of the tissue. The stromal vascular fraction was examined for isolation yield, viability, clonogenicity, and differentiation capacity.; The process of shuffling changed the macroscopic but not the microscopic structure of the lipoaspirated fat. No difference in cell number, viability, number of lipid droplets, vascular architecture, or ratio of cell composition was found. Analysis of the stromal vascular fraction, apart from large interdonor variability, did not show a significant change in isolation yield, viability, clonogenicity, or adipogenic differentiation capacity of the expanded cells.; The mechanical procedure of shuffling lipoaspirated fat does not alter its tissue viability or its microscopic structure. The absence of impact on the stromal vascular fraction in the assessed parameters suggests that shuffling can be executed according to surgical needs

Osteoblastic differentiation of Wharton jelly biopsy specimens and their mesenchymal stromal cells after serum-free culture

Cleft lip and cleft palate are increasingly being detected by prenatal ultrasound, which raises the opportunity of using the patient's own osteogenicity from umbilical cord mesenchymal cells for bony repair. The authors address the growth of the cells under a fully defined and regulated protocol.; Wharton jelly-derived mesenchymal stromal cells were isolated and expanded as a monolayer with defined serum-free medium. Osteoblastic differentiation was tested in the cells and in the entire Wharton jelly biopsy specimens. The serum-free-cultured cells were included in hydroxyapatite granule-fibrin constructs and, without predifferentiation, subcutaneously implanted into immunoincompetent mice.; Isolation and expansion of Wharton jelly-derived mesenchymal stromal cells were consistently successful under serum-free conditions, and the cells expressed standard mesenchymal stromal cell markers. The serum-free-cultivated cells produced a mineralized extracellular matrix under osteogenic differentiation, with a significant increase of osteoblastic lineage gene expression (Hox-A10 and Runx2) and an up-regulation of downstream osteogenic genes (OSX, OCN, ALPL, and BSP2). In vivo, they formed a dense matrix adjacent to the granules after 8 weeks, but no lamellar bone. serum-free-cultivated entire Wharton jelly biopsy specimens produced a mineralized extracellular matrix within the collagen matrix of the Wharton jelly.; The osteogenic differentiation potential of Wharton jelly-derived mesenchymal stromal cells was maintained under serum-free isolation and expansion techniques. The cells without predifferentiation form a dense collagen matrix but not bone in vivo. Moreover, entire Wharton jelly biopsy specimens showed periosteal-like mineralization under osteogenic differentiation, which offers new options for autologous bone tissue engineering, including cleft palate surgery

The regeneration potential after human and autologous stem cell transplantation in a rat sciatic nerve injury model can be monitored by MRI

Traumatic nerve injuries are a major clinical challenge. Tissue engineering using a combination of nerve conduits and cell-based therapies represents a promising approach to nerve repair. The aim of this study was to examine the regeneration potential of human adipose-derived stem cells (hASCs) after transplantation in a nonautogenous setting and to compare them with autogenous rat ASCs (rASCs) for early peripheral nerve regeneration. Furthermore, the use of MRI to assess the continuous process of nerve regeneration was elaborated. The sciatic nerve injury model in female Sprague-Dawley rats was applied, and a 10-mm gap created by using a fibrin conduit seeded with the following cell types: rASCs, Schwann cell (SC)-like cells from rASC, rat SCs (rSCs), hASCs from the superficial and deep abdominal layer, as well as human stromal vascular fraction (1 × 10(6) cells). As a negative control group, culture medium only was used. After 2 weeks, nerve regeneration was assessed by immunocytochemistry. Furthermore, MRI was performed after 2 and 4 weeks to monitor nerve regeneration. Autogenous ASCs and SC-like cells led to accelerated peripheral nerve regeneration, whereas the human stem cell groups displayed inferior results. Nevertheless, positive trends could be observed for hASCs from the deep abdominal layer. By using a clinical 3T MRI scanner, we were able to visualize the graft as a small black outline and small hyperintensity indicating the regenerating axon front. Furthermore, a strong correlation was found between the length of the regenerating axon front measured by MRI and the length measured by immunocytochemistry (r = 0.74, p = 0.09). We successfully transplanted and compared human and autologous stem cells for peripheral nerve regeneration in a rat sciatic nerve injury model. Furthermore, we were able to implement the clinical 3T MRI scanner to monitor the efficacy of cellular therapy over time

Improved adipocyte viability in autologous fat grafting with ascorbic acid-supplemented tumescent solution

In reconstructive surgery, fat volume augmentation is often necessary for esthetic or functional reasons. As an alternative to synthetic and xenogeneic materials, autologous fat grafting (AFG) based on liposuction is gaining popularity, yet successful transplantation and long-term volume maintenance are difficult. Standard tumescent solution formulations neglect adipocyte and stromal vascular fraction (SVF) cell survival during extraction, as well as SVF differentiation into adipocytes thereafter, all of which are crucial for the success of AFG. Here we hypothesized that addition of ascorbic acid (AA) to the tumescent solution could prevent liposuction-induced cell damage

Peripheral Nerve Repair: Multimodal Comparison of the Long-Term Regenerative Potential of Adipose Tissue-Derived Cells in a Biodegradable Conduit

Tissue engineering is a popular topic in peripheral nerve repair. Combining a nerve conduit with supporting adipose-derived cells could offer an opportunity to prevent time-consuming Schwann cell culture or the use of an autograft with its donor site morbidity and eventually improve clinical outcome. The aim of this study was to provide a broad overview over promising transplantable cells under equal experimental conditions over a long-term period. A 10-mm gap in the sciatic nerve of female Sprague-Dawley rats (7 groups of 7 animals, 8 weeks old) was bridged through a biodegradable fibrin conduit filled with rat adipose-derived stem cells (rASCs), differentiated rASCs (drASCs), human (h)ASCs from the superficial and deep abdominal layer, human stromal vascular fraction (SVF), or rat Schwann cells, respectively. As a control, we resutured a nerve segment as an autograft. Long-term evaluation was carried out after 12 weeks comprising walking track, morphometric, and MRI analyses. The sciatic functional index was calculated. Cross sections of the nerve, proximal, distal, and in between the two sutures, were analyzed for re-/myelination and axon count. Gastrocnemius muscle weights were compared. MRI proved biodegradation of the conduit. Differentiated rat ASCs performed significantly better than undifferentiated rASCs with less muscle atrophy and superior functional results. Superficial hASCs supported regeneration better than deep hASCs, in line with published in vitro data. The best regeneration potential was achieved by the drASC group when compared with other adipose tissue-derived cells. Considering the ease of procedure from harvesting to transplanting, we conclude that comparison of promising cells for nerve regeneration revealed that particularly differentiated ASCs could be a clinically translatable route toward new methods to enhance peripheral nerve repair

Implantation of Stromal Vascular Fraction Progenitors at Bone Fracture Sites: From a Rat Model to a First-in-Man Study

Stromal Vascular Fraction (SVF) cells freshly isolated from adipose tissue include osteogenic- and vascular-progenitors, yet their relevance in bone fracture healing is currently unknown. Here, we investigated whether human SVF cells directly contribute to the repair of experimental fractures in nude rats, and explored the feasibility/safety of their clinical use for augmentation of upper arm fractures in elderly individuals. Human SVF cells were loaded onto ceramic granules within fibrin gel and implanted in critical nude rat femoral fractures after locking-plate osteosynthesis, with cell-free grafts as control. After 8 weeks, only SVF-treated fractures did not fail mechanically and displayed formation of ossicles at the repair site, with vascular and bone structures formed by human cells. The same materials combined with autologous SVF cells were then used to treat low-energy proximal humeral fractures in 8 patients (64-84 years old) along with standard open reduction and internal fixation. Graft manufacturing and implantation were compatible with intraoperative settings and led to no adverse reactions, thereby verifying feasibility/safety. Biopsies of the repair tissue after up to 12 months, upon plate revision or removal, demonstrated formation of bone ossicles, structurally disconnected and morphologically distinct from osteoconducted bone, suggesting the osteogenic nature of implanted SVF cells. We demonstrate that SVF cells, without expansion or exogenous priming, can spontaneously form bone tissue and vessel structures within a fracture-microenvironment. The gained clinical insights into the biological functionality of the grafts, combined with their facile, intra-operative manufacturing modality, warrant further tests of effectiveness in larger, controlled trials. Stem Cells 2016;34:2956-2966

Dividing and conquering the fastest-growing genus: Towards a natural sectional classification of the mega-diverse genus Begonia (Begoniaceae)

The pantropical genus Begonia is the sixth-largest genus of flowering plants, including 1870 species. The sections of Begonia are used frequently as analogues to genera in other families but, despite their taxonomic utility, few of the current sections have been examined in the light of molecular phylogenetic analyses. We present herein the largest, most representative phylogeny of Begonia published to date and a subsequent provisional sectional classification of the genus. We utilised three plastid markers for 574 species and 809 accessions of Begonia and used Hillebrandia as an outgroup to produce a dated phylogeny. The relationships between some species and sections are poorly resolved, but many sections and deeper nodes receive strong support. We recognise 70 sections of Begonia including 5 new sections: Astrothrix, Ephemera, Jackia, Kollmannia, and Stellandrae; 4 sections are reinstated from synonymy: Australes, Exalabegonia, Latistigma and Pereira; and 5 sections are newly synonymised. The new sectional classification is discussed with reference to identifying characters and previous classifications

Dividing and conquering the fastest-growing genus: Towards a natural sectional classification of the mega-diverse genus Begonia (Begoniaceae)

The pantropical genus Begonia is the sixth-largest genus of flowering plants, including 1870 species. The sections of Begonia are used frequently as analogues to genera in other families but, despite their taxonomic utility, few of the current sections have been examined in the light of molecular phylogenetic analyses. We present herein the largest, most representative phylogeny of Begonia published to date and a subsequent provisional sectional classification of the genus. We utilised three plastid markers for 574 species and 809 accessions of Begonia and used Hillebrandia as an outgroup to produce a dated phylogeny. The relationships between some species and sections are poorly resolved, but many sections and deeper nodes receive strong support. We recognise 70 sections of Begonia including 5 new sections: Astrothrix, Ephemera, Jackia, Kollmannia, and Stellandrae; 4 sections are reinstated from synonymy: Australes, Exalabegonia, Latistigma and Pereira; and 5 sections are newly synonymised. The new sectional classification is discussed with reference to identifying characters and previous classifications

Tannin phenotyping of the Vitaceae reveals a phylogenetic linkage of epigallocatechin in berries and leaves

International audienceBackground and AimsCondensed tannins, responsible for berry and wine astringency, may have been selected during grapevine domestication (Narduzzi et al., 2015). This work examines the phylogenetic distribution of condensed tannins throughout the Vitaceae phylogenetic tree.MethodsGreen berries and mature leaves of representative true-to-type members of the Vitaceae were collected before “véraison”, freeze-dried, pulverised, and condensed tannins measured following depolymerization by nucleophilic addition of 2-mercaptoethanol to the C4 of the flavan-3-ol units in an organic acidic medium. Reaction products were separated and quantitated by UPLC/DAD/MS.Key Results and ConclusionsThe original ability to incorporate epigallocatechin (EGC) into grapevine condensed tannins was lost independently in both the American and Eurasian/Asian branches of the Vitaceae, with exceptional cases of reversion to the ancestral EGC phenotype. This is particularly true in the genus Vitis, where we now find two radically distinct groups differing with respect to EGC content. While Vitis species from Asia are void of EGC, 50% of the New World Vitis harbour EGC. Interestingly, the presence of EGC is tightly coupled with the degree of leaf margin serration. Noticeably, the rare Asian EGC-forming species are phylogenetically close to Vitis vinifera, the only remnant representative of Vitis in Eurasia. Both the wild ancestral V. vinifera subsp. sylvestris as well as the domesticated V. vinifera subsp. sativa can accumulate EGC and activate galloylation biosynthesis that compete for photoassimilates and reductive power