An injectable non-cross-linked hyaluronic-acid gel containing therapeutic spheroids of human adipose-derived stem cells.

For chronic wounds, the delivery of stem cells in spheroidal structures can enhance graft survival and stem cell potency. We describe an easy method for the 3D culture of adipose-derived stem/stromal cells (ASCs) to prepare a ready-to-use injectable. We transferred suspensions of monolayer-cultured ASCs to a syringe containing hyaluronic acid (HA) gel, and then incubated the syringe as a 3D culture vessel. Spheroids of cells formed after 12 h. We found that 6 × 106 ASCs/ml in 3% HA gel achieved the highest spheroid density with appropriate spheroid sizes (20-100 µm). Immunocytology revealed that the stem cell markers, NANOG, OCT3/4, SOX-2, and SSEA-3 were up-regulated in the ASC spheroids compared with those in nonadherent-dish spheroids or in monolayer cultured ASCs. In delayed wound healing mice models, diabetic ulcers treated with ASC spheroids demonstrated faster wound epithelialization with thicker dermis than those treated with vehicle alone or monolayer cultured ASCs. In irradiated skin ulcers in immunodeficient mice, ASC spheroids exhibited faster healing and outstanding angiogenic potential partly by direct differentiation into α-SMA+ pericytes. Our method of 3D in-syringe HA gel culture produced clinically relevant amounts of ready-to-inject human ASC microspheroids that exhibited superior stemness in vitro and therapeutic efficacy in pathological wound repair in vivo.

Therapeutic Potential of Human Adipose-Derived Stem/Stromal Cell Microspheroids Prepared by Three-Dimensional Culture in Non-Cross-Linked Hyaluronic Acid Gel.

UNLABELLED: Three-dimensional culture of mesenchymal stem/stromal cells for spheroid formation is known to enhance their therapeutic potential for regenerative medicine. Spheroids were prepared by culturing human adipose-derived stem/stromal cells (hASCs) in a non-cross-linked hyaluronic acid (HA) gel and compared with dissociated hASCs and hASC spheroids prepared using a nonadherent dish. Preliminary experiments indicated that a 4% HA gel was the most appropriate for forming hASC spheroids with a relatively consistent size (20-50 µm) within 48 hours. Prepared spheroids were positive for pluripotency markers (NANOG, OCT3/4, and SOX-2), and 40% of the cells were SSEA-3-positive, a marker of the multilineage differentiating stress enduring or Muse cell. In contrast with dissociated ASCs, increased secretion of cytokines such as hepatocyte growth factor was detected in ASC spheroids cultured under hypoxia. On microarray ASC spheroids showed upregulation of some pluripotency markers and downregulation of genes related to the mitotic cell cycle. After ischemia-reperfusion injury to the fat pad in SCID mice, local injection of hASC spheroids promoted tissue repair and reduced the final atrophy (1.6%) compared with that of dissociated hASCs (14.3%) or phosphate-buffered saline (20.3%). Part of the administered hASCs differentiated into vascular endothelial cells. ASC spheroids prepared in a HA gel contain undifferentiated cells with therapeutic potential to promote angiogenesis and tissue regeneration after damage. SIGNIFICANCE: This study shows the therapeutic value of human adipose-derived stem cell spheroids prepared in hyarulonic acid gel. The spheroids have various benefits as an injectable cellular product and show therapeutic potential to the stem cell-depleted conditions such as diabetic chronic skin ulcer.

Micronized cellular adipose matrix as a therapeutic injectable for diabetic ulcer.

BACKGROUND: Despite the clinical potential of adipose-derived stem/stromal cells (ASCs), there are some clinical difficulties due to the regulation of cell therapies. MATERIALS & METHODS: Micronized cellular adipose matrix (MCAM) injectable was prepared through selective extraction of connective tissue fractions in fat tissue only through mechanical minimal manipulation procedures. RESULTS: It retained some capillaries and ASCs, but most adipocytes were removed. The presence of viable ASCs, vascular endothelial cells was confirmed and ASCs of MCAM kept intact mesenchymal differentiation capacity. In diabetic mice, skin wounds treated with MCAM showed significantly accelerated healing compared with phosphate-buffered saline-treated ones. CONCLUSION: The proven potential of MCAM to accelerate healing in ischemic diabetic ulcers may offer a simple, safe and minimally invasive means for tissue repair and revitalization.

Condensation of tissue and stem cells for fat grafting.

Aspirated fat contains unnecessary components such as water, oil, and blood cells. For better outcomes, tissue purification and condensation are useful, especially when injection volume to the recipient site is limited. Because aspirated fat is relatively poor in adipose-derived stem/stromal cells (ASCs), ASC condensation seems important for obtaining better regeneration and retention. Reducing tissue volume by removing some adipocytes or supplementation of stromal vascular fraction or ASCs can increase the ASC/adipocyte ratio in the graft. Clinical results of ASC supplementation remain controversial, but ASC condensation seems to lead to expanding applications of fat grafting into revitalization of stem cell-depleted tissue.

Differential contributions of graft-derived and host-derived cells in tissue regeneration/remodeling after fat grafting.

BACKGROUND: Recent research indicates that the adipose tissue of nonvascularized grafts is completely remodeled within 3 months, although origins of next-generation cells are unclear. METHODS: Inguinal fat pads of green fluorescent protein mice and wild-type mice were cross-transplanted beneath the scalp. At 1, 2, 4, and 12 weeks after transplantation, grafted fat was harvested, weighed, and analyzed through immunohistochemistry, whole-mount staining, and flow cytometry of cell isolates. Bone marrow of green fluorescent protein mice was transplanted to wild-type mice (after irradiation). Eight weeks later, these mice also received fat grafts, which were analyzed as well. RESULTS: The majority of host-derived cells detected during remodeling of grafted fat were macrophages (>90 percent at the early stage; 60 percent at 12 weeks). Cell origins were analyzed at 12 weeks (i.e., when completely regenerated). At this point, mature adipocytes were largely derived from adipose-derived stem/stromal cells of grafts. Although vascular wall constituents were chiefly graft derived, vascular endothelial cells originated equally from graft and host bone marrow. Adipose-derived stem/stromal cells of regenerated fat were an admixture of grafted, host nonbone marrow, and host bone marrow cells. CONCLUSIONS: The above findings underscore the importance of adipose stem/stromal cells in the grafted fat for adipocyte regeneration. Host bone marrow and local tissues contributed substantially to capillary networks and provided new adipose-derived stem/stromal cells. An appreciation of mechanisms that are operant in this setting stands to improve clinical outcomes of fat grafting and cell-based therapies.

Therapeutic Potential of Adipose-Derived SSEA-3-Positive Muse Cells for Treating Diabetic Skin Ulcers.

Stage-specific embryonic antigen-3 (SSEA-3)-positive multipotent mesenchymal cells (multilineage differentiating stress-enduring [Muse] cells) were isolated from cultured human adipose tissue-derived stem/stromal cells (hASCs) and characterized, and their therapeutic potential for treating diabetic skin ulcers was evaluated. Cultured hASCs were separated using magnetic-activated cell sorting into positive and negative fractions, a SSEA-3+ cell-enriched fraction (Muse-rich) and the remaining fraction (Muse-poor). Muse-rich hASCs showed upregulated and downregulated pluripotency and cell proliferation genes, respectively, compared with Muse-poor hASCs. These cells also released higher amounts of certain growth factors, particularly under hypoxic conditions, compared with Muse-poor cells. Skin ulcers were generated in severe combined immunodeficiency (SCID) mice with type 1 diabetes, which showed delayed wound healing compared with nondiabetic SCID mice. Treatment with Muse-rich cells significantly accelerated wound healing compared with treatment with Muse-poor cells. Transplanted cells were integrated into the regenerated dermis as vascular endothelial cells and other cells. However, they were not detected in the surrounding intact regions. Thus, the selected population of ASCs has greater therapeutic effects to accelerate impaired wound healing associated with type 1 diabetes. These cells can be achieved in large amounts with minimal morbidity and could be a practical tool for a variety of stem cell-depleted or ischemic conditions of various organs and tissues.

Application of normobaric hyperoxygenation to an ischemic flap and a composite skin graft.

BACKGROUND: Hyperbaric oxygenation has been used for various purposes, but its clinical application is limited due to its pulmonary toxicity. We evaluated the therapeutic value of normobaric hyperoxygenation (NBO) for vascularized and nonvascularized tissue transplantation. METHODS: Tissue oxygen partial pressure (PtO2) was measured for various organs in mice under inspiratory oxygen of 20%, 60%, or 100%. A rectangular skin flap (1 × 4 cm) or a composite skin graft (2 × 2 cm) was made on the back of mice, which were housed under 20% or 60% oxygen for the first 3 days after surgery. Cell survival was also examined in organ culture skin samples. RESULTS: PtO2 varied among tissues/organs, but increased depending on inspiratory oxygen concentration in all tissues/organs. Although NBO with 100% O2 was toxic, NBO with 60% O2 was safe even when used continuously for a long period. NBO did not significantly improve survival of the rectangular skin flap. On the other hand, in the composite skin graft model, the engraftment area increased significantly (52 ± 10 at 20% vs 68 ± 5.1 at 60%) and contraction decreased significantly (42 ± 8.0 at 20% vs 27 ± 5.7 at 60%). Organ culture of a composite skin sample showed significant cell death under lower oxygen concentrations, supporting the data in vivo. CONCLUSIONS: The composite graft was maintained until revascularization by plasmatic diffusion from surrounding tissues, in which PtO2 was improved by NBO. NBO may be an effective adjunct therapy that can be performed readily after nonvascularized tissue grafting.

Normobaric hyperoxygenation enhances initial survival, regeneration, and final retention in fat grafting.

BACKGROUND: Fat grafting is a promising modality for soft-tissue augmentation/reconstruction. However, grafted fat tissue is not initially perfused and relies on plasmatic diffusion from the recipient bed until revascularization occurs. The authors evaluated the therapeutic effects of normobaric hyperoxygenation for enhancing fat graft retention. METHODS: Aspirated human fat tissue was cultured under tissue hypoxia (1% oxygen), normoxia (6%), and hyperoxia (20%) levels, and evaluated for adipocyte viability. Inguinal fat pads were autografted under mouse scalps (n=36), and mice were housed in either 20% (control) or 60% (normobaric hyperoxygenation) atmospheric oxygen for the first 3 days, and then returned to normoxia. Samples harvested at 0, 1, 2, 4, 8, and 12 weeks were analyzed immunohistochemically for adipocyte viability and regeneration. RESULTS: Organ culture adipocytes died more quickly under lower oxygen tensions; thus, hyperoxygenation of recipient tissues may delay adipocyte death after fat grafting. Autografted mouse adipose tissue underwent dynamic remodeling, from ischemic degeneration to partial regeneration, over 12 weeks. Normobaric hyperoxygenation grafted samples showed significantly larger survival zones and engraftment scores (calculated using sample weight and adipocyte viability) at 1 and 12 weeks, respectively, than control samples. In addition, adipocyte regeneration (number of perilipin-positive preadipocytes), which peaked at 4 weeks, was significantly increased in normobaric hyperoxygenation samples. CONCLUSION: The normobaric hyperoxygenation protocol using 60% oxygen can be safely applied to enhance adipocyte survival, regeneration, and final engraftment after fat grafting.

Chronic inflammation and progressive calcification as a result of fat necrosis: the worst outcome in fat grafting.

BACKGROUND: Autologous fat injection into the breast has been performed widely for breast augmentation and reconstruction because of recent technical and scientific advancements. However, it is important to learn what occurs and how problematic it can be if fat grafting is not conducted appropriately. METHODS: Oil cysts were explanted from three subjects who underwent cosmetic fat grafting to the breast 2, 4, and 6 years previously. The oil cyst samples were examined histopathologically. Computed tomographic, magnetic resonance imaging, and mammographic images obtained sequentially after fat grafting were also analyzed. RESULTS: The cyst wall consisted of innermost and outermost fibrous layers and intermediate tissue that contained the regular adipose portion, a degenerated adipose portion, and a fibrous area. Eggshell-like macrocalcifications were seen in the inner surface. Numerous inflammatory cells, mainly MAC2/CD206 anti-inflammatory M2 macrophages, were observed in the degenerated adipose portion. Oil cysts with a longer history showed more calcifications in the innermost layer and a larger fibrous area adjacent to the degenerated fat portion than those with a shorter history. These histopathologic findings and clinical computed tomographic images revealed that oil cysts continued to be inflammatory and calcifications continued to develop over several years. CONCLUSIONS: After fat necrosis, long-term chronic inflammation persists and calcification seems to progress without limits. Oil cysts are the worst outcome of fat grafting and must be avoided by standardizing meticulous injection techniques. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, V.

Degeneration, regeneration, and cicatrization after fat grafting: dynamic total tissue remodeling during the first 3 months.

BACKGROUND: Fat grafting is promising, but clinical outcomes are not always predictable. The mechanisms of tissue revascularization/regeneration, and tissue necrosis and subsequent absorption/fibrosis of the graft, are poorly understood. METHODS: An autologous inguinal fat pad was transplanted under the scalp of mice, and detailed cellular events during the first 3 months were investigated with immunohistochemistry. RESULTS: Except for the most superficial surviving zone, death of all adipocytes was confirmed at 1 week. Perilipin-positive small new adipocytes appeared at 1 week and peaked in number at 4 weeks in the regenerating zone (the second zone). In the most central necrotizing zone, adipogenesis did not occur and many inflammatory cells were observed after 2 weeks. CD34+/Ki67+ proliferating adipose stem/progenitor cells were seen at 1 to 4 weeks, but the majority of proliferating cells were MAC2+ monocytes/macrophages. Although CD206+ M1 macrophages surrounded oil droplets for phagocytosis, CD206+ M2 macrophages appeared in areas where adipocyte replacement failed and formed multiple layers for cicatrization of oil drop spaces. Adipogenesis was complete by 12 weeks, but stabilization of nonregenerated areas was still ongoing at that time. Lipid droplets derived from dead adipocytes were absorbed slowly and thus aided adipose remodeling by maintaining the space until adipocyte regeneration. CONCLUSIONS: Dynamic remodeling after fat grafting was confirmed. Adipocyte fate differed, depending on the microenvironment: intact survival, replacement with a new adipocyte, or replacement with cicatrization/oil cyst. This detailed understanding will help refine surgical grafting procedures and postoperative evaluation.

Enrichment isolation of adipose-derived stem/stromal cells from the liquid portion of liposuction aspirates with the use of an adherent column.

BACKGROUND AIMS: Adipose-derived stem/progenitor cells (ASCs) are typically obtained from the lipoaspirates; however, a smaller number of ASCs can be isolated without enzymatic digestion from the infranatant liposuction aspirate fluid (LAF). We evaluated the effectiveness of an adherent column, currently used to isolate mesenchymal stromal cells from bone marrow, to isolate LAF cells. METHODS: We applied peripheral blood (PB), PB mixed with cultured ASCs (PB-ASC), and LAF solution to the column and divided it into two fractions, the adherent (positive) and the non-adherent (negative) fractions. We compared this method with hypotonic hemolysis (lysis) for the red blood cell count, nucleated cells count and cell compositions as well as functional properties of isolated mesenchymal cells. RESULTS: The column effectively removed red blood cells, though the removal efficiency was slightly inferior to hemolysis. After column processing of PB-ASC, 60.5% of ASCs (53.2% by lysis) were selectively collected in the positive fraction, and the negative fraction contained almost no ASCs. After processing of LAF solution, nucleated cell yields were comparable between the column and hemolysis; however, subsequent adherent culture indicated that a higher average ASC yield was obtained from the column-positive samples than from the lysis samples, suggesting that the column method may be superior to hemolysis for obtaining viable ASCs. Mesenchymal differentiation and network formation assays showed no statistical differences in ASC functions between the lysis and column-positive samples. CONCLUSIONS: Our results suggest that a column with non-woven rayon and polyethylene fabrics is useful for isolating stromal vascular fraction cells from LAF solutions for clinical applications.

Cell and Tissue Damage after Skin Exposure to Ionizing Radiation: Short- and Long-Term Effects after a Single and Fractional Doses.

Ionizing radiation is often used to treat progressive neoplasms. However, the consequences of long-term radiation exposure to healthy skin tissue are poorly understood. We aimed to evaluate the short- and long-term radiation damage to healthy skin of the same irradiation given either as single or fractional doses. C57BL/J6 mice were randomly assigned to one of three groups: a control and two exposure groups (5 Gy ×2 or 10 Gy ×1). The inguinal area was irradiated (6-MeV beam) 1 week after depilation in the treatment groups. Skin samples were evaluated macroscopically and histologically for up to 6 months after the final exposure. After anagen hair follicle injury by irradiation, hair cycling resumed in both groups, but hair graying was observed in the 10 Gy ×1 group but not in the 5 Gy ×2 group, suggesting the dose of each fractional exposure is more relevant to melanocyte stem cell damage than the total dose. On the other hand, in the long term, the fractional double exposures induced more severe atrophy and capillary reduction in the dermis and subcutis, suggesting fractional exposure may cause more depletion of tissue stem cells and endothelial cells in the tissue. Thus, our results indicated that there were differences between the degrees of damage that occurred as a result of a single exposure compared with fractional exposures to ionizing radiation: the former induces more severe acute injury to the skin with irreversible depigmentation of hairs, while the latter induces long-term damage to the dermis and subcutis.

N-Cadherin is a prospective cell surface marker of human mesenchymal stem cells that have high ability for cardiomyocyte differentiation.

Mesenchymal stem cells (MSCs) are among the most promising sources of stem cells for regenerative medicine. However, the range of their differentiation ability is very limited. In this study, we explored prospective cell surface markers of human MSCs that readily differentiate into cardiomyocytes. When the cardiomyogenic differentiation potential and the expression of cell surface markers involved in heart development were analyzed using various immortalized human MSC lines, the MSCs with high expression of N-cadherin showed a higher probability of differentiation into beating cardiomyocytes. The differentiated cardiomyocytes expressed terminally differentiated cardiomyocyte-specific markers such as α-actinin, cardiac troponin T, and connexin-43. A similar correlation was observed with primary human MSCs derived from bone marrow and adipose tissue. Moreover, N-cadherin-positive MSCs isolated with N-cadherin antibody-conjugated magnetic beads showed an apparently higher ability to differentiate into cardiomyocytes than the N-cadherin-negative population. Quantitative polymerase chain reaction analyses demonstrated that the N-cadherin-positive population expressed significantly elevated levels of cardiomyogenic progenitor-specific transcription factors, including Nkx2.5, Hand1, and GATA4 mRNAs. Our results suggest that N-cadherin is a novel prospective cell surface marker of human MSCs that show a better ability for cardiomyocyte differentiation.

Comparative characterization of stromal vascular cells derived from three types of vascular wall and adipose tissue.

Multipotent stem/progenitor cells localize perivascularly in many organs and vessel walls. These tissue-resident stem/progenitor cells differentiate into vascular endothelial cells, pericytes, and other mesenchymal lineages, and participate in physiological maintenance and repair of vasculatures. In this study, we characterized stromal vascular cells obtained through the explant culture method from three different vessel walls in humans: arterial wall (ART; >500 µm in diameter), venous wall (VN; >500 µm in diameter), and small vessels in adipose tissue (SV; arterioles and venules, <100 µm in diameter). These were examined for functionality and compared with adipose-derived stem/stromal cells (ASCs). All stromal vascular cells of different origins presented fibroblast-like morphology and we could not visually discriminate one population from another. Flow cytometry showed that the cultured population heterogeneously expressed a variety of surface antigens associated with stem/progenitor cells, but CD105 was expressed by most cells in all groups, suggesting that the cells generally shared the characteristics of mesenchymal stem cells. Our histological and flow cytometric data suggested that the main population of vessel wall-derived stromal vascular cells were CD34(+)/CD31(-) and came from the tunica adventitia and areola tissue surrounding the adventitia. CD271 (p75NTR) was expressed by the vasa vasorum in the VN adventitia and by a limited population in the adventitia of SV. All three populations differentiated into multiple lineages as did ASCs. ART cells induced the largest quantity of calcium formation in the osteogenic medium, whereas ASCs showed the greatest adipogenic differentiation. SV and VN stromal cells had greater potency for network formation than did ART stromal cells. In conclusion, the three stromal vascular populations exhibited differential functional properties. Our results have clinical implications for vascular diseases such as arterial wall calcification and possible applications to regenerative therapies involving each vessel wall-resident stromal population.

Stromal vascular fraction isolated from lipo-aspirates using an automated processing system: bench and bed analysis.

The heterogeneous stromal vascular fraction (SVF), containing adipose-derived stem/progenitor cells (ASCs), can be easily isolated through enzymatic digestion of aspirated adipose tissue. In clinical settings, however, strict control of technical procedures according to standard operating procedures and validation of cell-processing conditions are required. Therefore, we evaluated the efficiency and reliability of an automated system for SVF isolation from adipose tissue. SVF cells, freshly isolated using the automated procedure, showed comparable number and viability to those from manual isolation. Flow cytometric analysis confirmed an SVF cell composition profile similar to that after manual isolation. In addition, the ASC yield after 1 week in culture was also not significantly different between the two groups. Our clinical study, in which SVF cells isolated with the automated system were transplanted with aspirated fat tissue for soft tissue augmentation/reconstruction in 42 patients, showed satisfactory outcomes with no serious side-effects. Taken together, our results suggested that the automated isolation system is as reliable a method as manual isolation and may also be useful in clinical settings. Automated isolation is expected to enable cell-based clinical trials in small facilities with an aseptic room, without the necessity of a good manufacturing practice-level cell processing area.

Characterization of human adipose tissue-resident hematopoietic cell populations reveals a novel macrophage subpopulation with CD34 expression and mesenchymal multipotency.

Adipose tissue (AT) is composed of mature adipocytes and stromal vascular fraction (SVF) cells, including adipose stem/stromal cells (ASCs). We characterized hematopoietic cells residing in human nonobese AT by analyzing the SVF isolated from human lipoaspirates and peripheral blood (PB). Flow cytometry revealed that AT-resident hematopoietic cells consisted of AT-resident macrophages (ATMs) or lymphocytes with a negligible number of granulocytes. AT-resident lymphocytes were composed of helper T cells and natural killer cells. Almost no B cells and few cytotoxic T cells were observed in nonobese AT. More than 90% of ATMs were M2 state CD206(+) macrophages (CD45(+)/CD14(+)) that were located in the periendothelium or interstitial spaces between adipocytes. We also discovered a novel subpopulation of CD34(+)/CD206(+) ATMs (11.1% of CD206(+)ATMs) that localized in the perivascular region. Microarray of noncultured CD34(+)/CD206(+) ATMs, CD34(-)/CD206(+) ATMs, CD45(-)/CD31(-)/CD34(+) ASCs, and PB-derived circulating monocytes revealed that CD34(+)/CD206(+) ATMs shared characteristics with ASCs and circulating monocytes. Unlike CD34(-)/CD206(+) ATMs, CD34(+)/CD206(+) ATMs could grow in adherent culture and were capable of differentiating into multiple mesenchymal (adipogenic, osteogenic, and chondrogenic) lineages, similar to ASCs. CD34(+)/CD206(+) ATMs grew rapidly and lost expression of CD45, CD14, and CD206 by passage 3, which resulted in a similar expression profile to ASCs. Thus, this novel ATM subpopulation (CD45(+)/CD14(+)/CD34(+)/CD206(+)) showed distinct biological properties from other ATMs and circulating monocytes/macrophages. The CD34(+)/CD206(+) ATMs possessed characteristics similar to ASCs, including adherence, localization, morphology, and mesenchymal multipotency. This AT-resident subpopulation may have migrated from the bone marrow and may be important to tissue maintenance and remolding.

Semipermanent volumization by an absorbable filler: onlay injection technique to the bone.

BACKGROUND: Hyaluronic acid (HA) fillers have become the most popular tool for wrinkle treatment and volumization, although HA is generally absorbed within 6-12 months and requires repeated treatments to maintain the effects. METHODS: HA was injected onto the bone for volumization with a small 30-gauge needle to examine the long-lasting effects. Of the 63 Japanese patients with 97 treated sites followed up more than 12 months, 51 had HA injections for cosmetic purposes and 12 were treated for reconstructive volumization of facial deformity such as localized scleroderma and postsurgical bony deformity. Treated sites included the forehead, temple, nasal root, mentum, tear trough, and infraorbital sulcus. RESULTS: After long-term follow-up (12-93 months, mean = 21.6), persistent volumizing effects were observed in most patients. In fact, 86.6% of the treated sites showed >50% volume retention and 49.5% showed >75% retention. Magnetic resonance imaging analyses revealed that the injected space was well maintained, capsulated, and filled with heterogeneous content. Magnetic resonance imaging quantitative T2 maps indicated that much of the injected HA was replaced with other materials. Together with clinical inspection, these findings suggest that onlay injection of HA on the bone induced formation of capsule, fibrosis, and/or calcification/ossification, which contributed to persistent volumization. CONCLUSIONS: Semipermanent volumizing effects can be achieved by HA injection if the target area has an underlying bony floor. Periosteal stem cells may be activated by HA injection and may contribute to persistent volumizing effects. This treatment may be a much less invasive alternative to fat or bone grafting.

The fate of adipocytes after nonvascularized fat grafting: evidence of early death and replacement of adipocytes.

BACKGROUND: Clinical outcomes following fat grafting are variable and technique dependent, and it is unknown how the graft is revascularized. The authors recently observed that living and dead adipocytes can be differentiated not with hematoxylin and eosin staining but with immunohistochemistry for perilipin. METHODS: The viability of cellular components (adipocytes, adipose stem/stromal/progenitor cells, vascular endothelial cells, and hematopoietic cells) in human adipose tissue was evaluated using (1) stored lipoaspirates, (2) cultured cells, and (3) organ-cultured adipose tissue. In addition, the groin fat pad (150 to 200 mg) in mice was transplanted under the scalp, and the graft was stained at 0, 1, 2, 3, 5, 7, or 14 days. RESULTS: In vitro studies revealed that adipocytes are most susceptible to death under ischemic conditions, although adipose-derived stromal cells can remain viable for 3 days. The in vivo study indicated that most adipocytes in the graft began to die on day 1, and only some of the adipocytes located within 300 µm of the tissue edge survived. The number of proliferating cells increased from day 3, and an increase in viable adipocyte area was detected from day 7, suggesting that repair/regeneration of the dead tissue had begun. CONCLUSIONS: The authors show convincing evidence of very dynamic remodeling of adipose tissue after nonvascularized grafting. The authors observed three zones from the periphery to the center of the graft: the surviving area (adipocytes survived), the regenerating area (adipocytes died, adipose-derived stromal cells survived, and dead adipocytes were replaced with new ones), and the necrotic area (both adipocytes and adipose-derived stromal cells died).

Therapeutic potential of fibroblast growth factor-2 for hypertrophic scars: upregulation of MMP-1 and HGF expression.

Although hypertrophic scars (HTSs) and keloids are challenging problems, their pathogenesis is not well understood, making therapy difficult. We showed that matrix metalloproteinase (MMP)-1 expression was downregulated in HTS compared with normal skin from the same patients, whereas type 1 and 3 collagen and transforming growth factor-ß (TGF-ß) were upregulated. These differences, however, were not seen in cultured fibroblasts, suggesting the involvement of microenvironmental factors in the pathogenesis of HTS. Fibroblast growth factor-2 (FGF-2) highly upregulated the expression of MMP-1 and hepatocyte growth factor (HGF) in both HTS-derived and control fibroblasts; the upregulation was reversed by extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) inhibitors. An animal study using human HTS tissue implanted into nude mice indicated that controlled-release FGF-2 resulted in significantly less weight and decreased hydroxyproline content in HTS. Degradation of collagen fibers in FGF-2-treated HTS was also confirmed histologically. Western blotting showed that FGF-2-treated HTS expressed significantly higher MMP-1 protein than control. Decreased MMP-1 expression may be an important transcriptional change in HTS, and its reversal as well as upregulation of HGF by FGF-2 could be a new therapeutic approach for HTS.