Composite bone graft of CaO-MgO-SiO2 glass-ceramics and CaSO4 ceramics for boosting bone formation rate.

This study develops a composite bone graft of CaO-MgO-SiO2 glass-ceramic and CaSO4 [abbreviated as (CMS)3-x(CS)x] via the sponge replication technique with weight fractions of x = 0, 1, 1.5, 2, and 3. The (CMS)1.5(CS)1.5 composite displays a superior degradability and, a suitable compressive strength of ∼3 MPa, and excellent cell proliferation and differentiation. The in vivo rat femur test in the hybrid-pore (CMS)1.5(CS)1.5 composite granules achieves a higher rate of bone formation, which is ∼2.7 times better than that of the commercial HAP/ß-TCP at 12 weeks. Improved expressions of osteocyte and mature osteocyte marker genes, namely (Spp1, Dmp1, and Fgf23), were observed in the (CMS)1.5(CS)1.5 group, indicating a faster differentiation into mature bone tissue. The ions release of (CMS)1.5(CS)1.5 through the ERK1/2 signaling pathway promotes osteogenic differentiation. The high bone generation rate can be attributed to faster active ions release and modified surface topography. This work highlights an excellent bone graft candidate for clinical applications in orthopedic surgery.

Delivery and Transcriptome Assessment of an In Vitro Three-Dimensional Proximal Tubule Model Established by Human Kidney 2 Cells in Clinical Gelatin Sponges.

The high prevalence of kidney diseases and the low identification rate of drug nephrotoxicity in preclinical studies reinforce the need for representative yet feasible renal models. Although in vitro cell-based models utilizing renal proximal tubules are widely used for kidney research, many proximal tubule cell (PTC) lines have been indicated to be less sensitive to nephrotoxins, mainly due to altered expression of transporters under a two-dimensional culture (2D) environment. Here, we selected HK-2 cells to establish a simplified three-dimensional (3D) model using gelatin sponges as scaffolds. In addition to cell viability and morphology, we conducted a comprehensive transcriptome comparison and correlation analysis of 2D and 3D cultured HK-2 cells to native human PTCs. Our 3D model displayed stable and long-term growth with a tubule-like morphology and demonstrated a more comparable gene expression profile to native human PTCs compared to the 2D model. Many missing or low expressions of major genes involved in PTC transport and metabolic processes were restored, which is crucial for successful nephrotoxicity prediction. Consequently, we established a cost-effective yet more representative model for in vivo PTC studies and presented a comprehensive transcriptome analysis for the systematic characterization of PTC lines.

In vivo vascularized scaffold with different shear-exposed models for lymphatic tissue regeneration.

Current clinical treatments on lymphedema provide promising results, but also result in donor site morbidities. The establishment of a microenvironment optimized for lymphangiogenesis can be an alternative way to enhance lymphatic tissue formation. Hemodynamic flow stimuli have been confirmed to have an influential effect on angiogenesis in tissue engineering, but not on lymphatic vessel formation. Here, the three in vivo scaffolds generated from different blood stimuli in the subcutaneous layer, in the flow through pedicle, and in an arterio-venous (AV) loop model, were created to investigate potential of lymphangiogenesis of scaffolds containing lymphatic endothelial cells (LECs). Our results indicated that AV loop model displayed better lymphangiogenesis in comparison to the other two models with slower flow or no stimuli. Other than hemodynamic force, the supplement of LECs is required for lymphatic vessel regeneration. The in vivo scaffold generated from AV loop model provides an effective approach for engineering lymphatic tissue in the clinical treatment of lymphedema.

Effects of Hyperbaric Oxygen Intervention on the Degenerated Intervertebral Disc: From Molecular Mechanisms to Animal Models.

MicroRNA (miRNA) 107 expression is downregulated but Wnt3a protein and ß-catenin are upregulated in degenerated intervertebral disc (IVD). We investigated mir-107/Wnt3a-ß-catenin signaling in vitro and in vivo following hyperbaric oxygen (HBO) intervention. Our results showed 96 miRNAs were upregulated and 66 downregulated in degenerated nucleus pulposus cells (NPCs) following HBO treatment. The 3' untranslated region (UTR) of the Wnt3a mRNA contained the "seed-matched-sequence" for miR-107. MiR-107 was upregulated and a marked suppression of Wnt3a was observed simultaneously in degenerated NPCs following HBO intervention. Knockdown of miR-107 upregulated Wnt3a expression in hyperoxic cells. HBO downregulated the protein expression of Wnt3a, phosphorylated LRP6, and cyclin D1. There was decreased TOP flash activity following HBO intervention, whereas the FOP flash activity was not affected. HBO decreased the nuclear translocation of ß-catenin and decreased the secretion of MMP-3 and -9 in degenerated NPCs. Moreover, rabbit serum KS levels and the stained area for Wnt3a and ß-catenin in repaired cartilage tended to be lower in the HBO group. We observed that HBO inhibits Wnt3a/ß-catenin signaling-related pathways by upregulating miR-107 expression in degenerated NPCs. HBO may play a protective role against IVD degeneration and could be used as a future therapeutic treatment.

Conjugation of bone grafts with NO-delivery dinitrosyl iron complexes promotes synergistic osteogenesis and angiogenesis in rat calvaria bone defects.

Craniofacial/jawbone deformities remain a significant clinical challenge in restoring facial/dental functions and esthetics. Despite the reported therapeutics for clinical bone tissue regeneration, the bioavailability issue of autografts and limited regeneration efficacy of xenografts/synthetic bone substitutes, however, inspire continued efforts towards functional conjugation and improvement of bioactive bone graft materials. Regarding the potential of nitric oxide (NO) in tissue engineering, herein, functional conjugation of NO-delivery dinitrosyl iron complex (DNIC) and osteoconductive bone graft materials was performed to optimize the spatiotemporal control over the delivery of NO and to activate synergistic osteogenesis and angiogenesis in rat calvaria bone defects. Among three types of biomimetic DNICs, [Fe2(µ-SCH2CH2COOH)2(NO)4] (DNIC-COOH) features a steady kinetics for cellular uptake by MC3T3-E1 osteoblast cells followed by intracellular assembly of protein-bound DNICs and release of NO. This steady kinetics for intracellular delivery of NO by DNIC-COOH rationalizes its biocompatibility and wide-spectrum cell proliferation effects on MC3T3-E1 osteoblast cells and human umbilical vein endothelial cells (HUVECs). Moreover, the bridging [SCH2CH2COOH]- thiolate ligands in DNIC-COOH facilitate its chemisorption to deproteinized bovine bone mineral (DBBM) and physisorption onto TCP (ß-tricalcium phosphate), respectively, which provides a mechanism to control the kinetics for the local release of loaded DNIC-COOH. Using rats with calvaria bone defects as an in vivo model, DNIC-DBBM/DNIC-TCP promotes the osteogenic and angiogenic activity ascribed to functional conjugation of osteoconductive bone graft materials and NO-delivery DNIC-COOH. Of importance, the therapeutic efficacy of DNIC-DBBM/DNIC-TCP on enhanced compact bone formation after treatment for 4 and 12 weeks supports the potential for clinical application to regenerative medicine.

Transcription Factor ATF3 Participates in DeltaNp63-Mediated Proliferation of Corneal Epithelial Cells.

Understanding the regulatory mechanisms underlying corneal epithelial cell (CEC) proliferation in vitro may provide the means to boost CEC production in cell therapy for ocular disorders. The transcription factor ΔNp63 plays a crucial role in the proliferation of CECs, but the underlying mechanisms is yet to be elucidated. TP63 and ΔNp63 are encoded by the TP63 gene via alternative promoters. We previously reported that both ΔNp63 and activating transcription factor (ATF3) are substantially expressed in cultured CECs, but the regulatory relationship between ΔNp63 and ATF3 is unknown. In the present study, we found that ΔNp63 increased ATF3 expression and ATF3 promoter activity in cultured CECs. The deletion of the p63 binding core site reduced ATF3 promoter activity. CECs overexpressing ATF3 exhibited significantly greater proliferation than control CECs. ATF3 knockdown suppressed the ΔNp63-induced increase in cell proliferation. Overexpression of ATF3 in CECs significantly elevated protein and mRNA levels of cyclin D. The protein levels of keratin 3/14, integrin ß1, and involucrin did not differ between ATF3-overexpressing CECs, ATF3-downregulated CECs, and control cells. In conclusion, our results suggest that ΔNp63 increases CEC proliferation via the ΔNp63/ATF3/CDK pathway.

The Impacts of Lymph on the Adipogenesis of Adipose-Derived Stem Cells.

BACKGROUND: The pathophysiology of adipose proliferation or differentiation in extremity lymphedema has not been thoroughly studied. This study investigated the impacts of the lymph harvested from lymphedematous limbs on the adipogenesis of adipose-derived stem cells (ASCs). METHODS: ASCs were isolated from the adipose tissue of normal extremities and cultured with lymph collected from Cheng lymphedema grade III to IV patients or adipogenic differentiation medium (ADM) and further subjected to differentiation and proliferation assay. The expression of adipogenesis genes was examined by real-time polymerase chain reaction to investigate the effect of lymph on ASCs. The level of adipogenic cytokines in the lymph was also evaluated. RESULTS: The adipocytes were significantly larger in lymphedema fat tissue compared with that in normal fat tissues ( P < 0.00). The adipogenesis of ASCs cultured in lymph was significantly enhanced compared with in ADM ( P = 0.008) on day 10, suggesting that the adipogenesis of ASCs was promoted under the lymph-cultured environment. The expression of adipogenesis genes, peroxisome proliferator-activated receptor ( P = 0.02), CAAT/enhancer-binding protein α ( P = 0.008); fatty-acid binding protein ( P = 0.004), and lipoprotein lipase ( P = 0.003), was statistically elevated when the ASCs were cultured with lymph. The insulin content in lymph was statistically higher in lymph ( P < 0.001) than in plasma. CONCLUSIONS: The adipogenesis of ASCs was promoted under the lymph-cultured environment with statistically increased adipogenesis genes of peroxisome proliferator-activated receptor, CAAT/enhancer-binding protein α, fatty-acid binding protein, and lipoprotein lipase. The excess lymph accumulated in the lymphedematous extremity contained a greater insulin/insulin-like growth factor-2. These adipogenic factors promoted the expression of early adipogenesis genes and led ASCs to undergo adipogenesis and differentiated into adipocytes. CLINICAL RELEVANCE STATEMENT: The accumulation of adipose tissue in the lymphedema region was contributed from the content of excess lymph.

Vascularized lymph node transplantation successfully reverses lymphedema and maintains immunity in a rat lymphedema model.

Vascularized lymph node transplantation (VLNT) has shown inspiring results for the treatment of lymphedema. Nevertheless, it remains unclear how VLNT restores lymphatic drainage and whether or not immunity recovers after surgery. Hindlimb lymphedema model was created using rats with extensive groin and popliteal lymph node removable following with radiotherapy, and the lymphedema was confirmed using indocyanine green (ICG) lymphangiography and micro-computer tomography for volume measurement. VLNT was performed 1 month later. Volume measurement, ICG lymphangiography, histology, and immune reaction were done 1 month after surgery. VLNT successfully reduced the volume of the lymphedema hindlimb, restored lymphatic drainage function with proven lymphatic channel, and reduced lymphedema-related inflammation and fibrosis. It promotes lymphangiogenesis shown from ICG lymphangiography, histology, and enhanced lymphangiogenesis gene expression. Dendritic cell trafficking via the regenerated lymphatic channels was successfully restored, and maintained systemic immune response was proved using dinitrofluorobenzene sensitization and challenge. VLNT effectively reduces lymphedema and promotes lymphatic regeneration in the capillary lymphatic but not the collecting lymphatic vessels. Along with the re-established lymphatic system was the restoration of immune function locally and systemically. This correlated to clinical experience regarding the reduction of swelling and infection episodes after VLNT in lymphedema patients.

Tracheal reconstruction with pedicled tandem grafts engineered by a radial stretch bioreactor.

The engineering of tracheal substitutes is pivotal in improving tracheal reconstruction. In this study, we aimed to investigate the effects of biomechanical stimulation on tissue engineering tracheal cartilage by mimicking the trachea motion through a novel radial stretching bioreactor, which enables to dynamically change the diameter of the hollow cylindrical implants. Applying our bioreactor, we demonstrated that chondrocytes seeded on the surface of Poly (ε-caprolactone) scaffold respond to mechanical stimulation by improvement of infiltration into implants and upregulation of cartilage-specific genes. Further, the mechanical stimulation enhanced the accumulation of cartilage neo-tissues and cartilage-specific extracellular macromolecules in the muscle flap-remodeled implants and reconstructed trachea. Nevertheless, the invasion of fibrous tissues in the reconstructed trachea was suppressed upon mechanical loading.

Tracheal Reconstruction with the Scaffolded Cartilage Sheets in an Orthotopic Animal Model.

Tracheal reconstruction remains challenged in clinical. We aimed to fabricate scaffolded cartilage sheets with rigid and elastic supports for tracheal reconstruction. The chondrocyte cell infiltration activity was examined in poly-caprolactone sheet scaffolds with various thicknesses and pore sizes after seeding cells on the top surface of the sheet scaffolds. The expression of cartilage-related genes and accumulation of sulfated glycosaminoglycans were elevated in the cell-scaffold composites upon chondrogenic induction. The thicker cartilage sheets represented stronger mechanical properties than the thinner cartilage sheets. Two different cartilage sheets were orthotopically implanted into a trachea in a rabbit model for 2, 4, and 16 weeks. Cartilage-related sulfated glycosaminoglycans and type II collagen macromolecules were stably expressed in the tracheal implants. However, the invasive migration of fibrous tissue and profibrotic collagen fibers into cartilage implants and the peripheral space surrounding the implants were elevated in a time-dependent manner. At week 16 postimplantation, airway stenosis was noticed under the thicker sheet implants, but not the thinner implants, suggesting that the thinner (1 mm thick) scaffolded cartilage sheet was an optimal candidate for tracheal reconstruction in this study. Finally, cartilage sheets could be a reconstructive therapy candidate applied to reconstruct defects in the trachea and other tissues composed of cartilage. Impact statement Tissue engineering is a promising approach to generate biological substitutes. We aimed to develop cartilage sheets as tracheal prosthesis used in tracheal reconstruction or regional repairing in the animal model. The formation of microvessels and the dynamics of reepithelialization were monitored for 16 weeks in tracheal implants of the engineered cartilage sheets. In this study, it was demonstrated that the tissue-engineered cartilage sheets are potential substitutes applied in the reconstruction of the trachea and other tissues composed of cartilage tissue. The cartilage sheets were thought of as biomaterials for personalized regenerative medicine since the dimensions, thickness, and pore sizes of cartilage sheets were tunable to fit the lesions that need to be reconstructed.

Calcitriol and enamel matrix derivative differentially regulated cemento-induction and mineralization in human periodontal ligament-derived cells.

BACKGROUND: Alveolar bone and cementum share many biological and developmental similarities. The mineralizing effect of calcitriol has been previously reported. Yet, its cemento-inductivity has not been confirmed. This study evaluated the potential cemento-inductivity effect of calcitriol and enamel matrix derivative (EMD) on human periodontal ligament-derived cells (hPDLCs). METHODS: The hPDLCs obtained from extracted third molars or premolars were cultured with calcitriol, or EMD. Cementogenic gene expression was examined using real-time quantitative reverse transcription polymerase chain reaction. Expression analysis also included cementoblast-specific markers, cementum protein 1 (CEMP1), cementum attachment protein (CAP), and recently reported cementoblast-enriched genes, secreted frizzled related protein 1 (SFRP1), and Dickkopf-related protein 1 (DKK1). Mineralization capacities were evaluated by alkaline phosphatase (ALP) activity, Alizarin Red, and Von Kossa staining followed by scanning electron microscope imaging and element mapping. RESULTS: Among tested conditions, 10 nM calcitriol enhanced most cementogenic gene expression, transforming growth factor-ß1, bone morphogenetic proteins (BMP-2 and BMP-4), core-binding factor subunit alpha-1/Runt-related transcription factor 2, Type I collagen, ALP, bone sialoprotein, osteopontin), osteocalcin, CEMP1, and CAP, and Wnt signaling negative modulators, SFRP1 and DKK1, along with highest ALP activity and mineralization formation in hPDLCs. However, only moderate CEMP1 protein was observed. In contrast, EMD stimulated stronger CEMP1 and CAP protein, but presented weaker mineralization capacity, hinting at the possibility that strong stimulation of mineralization might dominate cemetogenic specific factors and vice versa. CONCLUSIONS: Calcitriol demonstrated not only great osteoinductivity, but also the potential to induce cementogenic gene expression by initiating hPDLC differentiation and promoting mineralization. Compared with calcitriol, EMD promoted cemento-inductivity in hPDLCs at a later time point via highly expressed CEMP1 and CAP protein, but with less mineralization. Thus, calcitriol and EMD could provide differential enhancement of cemento-induction and mineralization, likely acting at various differentiation stages.

The Effect of Negative Pressure on Wound Healing and Regeneration in Closed Incisions under High Tension: Evidence from Animal Studies and Clinical Experience.

Closed-incision negative-pressure wound therapy (iNPWT) is known to enhance wound healing and tissue regeneration. The main aim of the present study is to investigate its effectiveness on enhancing wound healing under tension. An animal study was designed using a swine model by removing a skin flap to create a wound that could be closed primarily under tension, and iNPWT was applied. The enhancement of angiogenesis, lymphangiogenesis, collagen deposition, and tissue proliferation with reduced inflammation by iNPWT was confirmed by histology. The effect of iNPWT was further verified in patients receiving a profunda artery perforator (PAP) free flap for breast reconstruction. iNPWT was applied on the transversely designed donor site in continuous mode for 7 days, in which the wound was always closed under tension. A significant improvement in off-bed time was noted with the application of iNPWT (4.6 ± 1.1st and 5.5 ± 0.8th postoperative days in the iNPWT and control groups, respectively, p = 0.028). The control group (without iNPWT treatment) presented more cases of poor wound healing in the acute (23.1% vs. 0%) and wound breakdown in the late (23.1% vs. 8.3%) stages. The treatment of closed incisions under tension with iNPWT clinically enhances wound healing and tissue regeneration and with histological evidence.

Application of dental stem cells in three-dimensional tissue regeneration.

Dental stem cells can differentiate into different types of cells. Dental pulp stem cells, stem cells from human exfoliated deciduous teeth, periodontal ligament stem cells, stem cells from apical papilla, and dental follicle progenitor cells are five different types of dental stem cells that have been identified during different stages of tooth development. The availability of dental stem cells from discarded or removed teeth makes them promising candidates for tissue engineering. In recent years, three-dimensional (3D) tissue scaffolds have been used to reconstruct and restore different anatomical defects. With rapid advances in 3D tissue engineering, dental stem cells have been used in the regeneration of 3D engineered tissue. This review presents an overview of different types of dental stem cells used in 3D tissue regeneration, which are currently the most common type of stem cells used to treat human tissue conditions.

Creation of a rat lymphedema model using extensive lymph node dissection and circumferential soft tissue resection: Is this a reliable model?

INTRODUCTION: The medical demand for lymphedema treatment is huge since the disease mechanism remains unclear, and management are difficult. Our purpose was to develop a reliable lymphedema model mimicking the clinical scenario and allows a microsurgical approach. MATERIALS AND METHODS: Male Lewis rats weighing 400 to 450 g were used to create lymphedema with groin and popliteal lymph node dissection and creation of 5 mm circumferential skin defect (n = 6). A skin incision was made and closed primarily for control group (n = 5). Evaluation included indocyanine green (ICG) lymphangiography 1 and 2 months postoperatively, volume difference between bilateral hindlimbs measured using micro-CT, and the skin was harvested for histological evaluation 2 months postoperatively. RESULTS: Larger volume differences present in the lymphedema group (17.50 ± 7.76 vs. 3.73 ± 2.66%, p < .05). ICG lymphangiography indicated dermal backflow only in the lymphedema group. Increased thickness of the epidermis was noted in lymphedema group (28.50 ± 12.61 µm vs. 15.10 ± 5.41 µm, p < .0001). More CD45+ (35.6 ± 26.68 vs. 2.8 ± 4.23 cells/high power field [HPF], p < .0001), CD3+ (38.39 ± 20.17 vs. 9.73 ± 8.62 cells/HPF, p < .0001), and CD4+ cell infiltration (11.7 ± 7.71 vs. 2.0 ± 2.67 cells/HPF, p < .0001) were observed in the lymphedema group. Collagen type I deposition was more in the lymphedema group (0.15 ± 0.06 vs. 0.07 ± 0.03, p < .0005). CONCLUSIONS: A rat lymphedema model was successfully established. The model can be applied in lymphedema related research.

Fate of Fat Grafting In Vivo and In Vitro: Does the Suction-Assisted Lipectomy Device Matter?

BACKGROUND: Recently, there has been increasing research interest in identifying the effect of liposuction procedures on fat graft survival in order to clarify whether different harvest techniques affect the quality of fat grafts. OBJECTIVES: The aim of this study was to investigate the effect of 2 liposuction methods on the survival and regeneration potential of grafted fat tissue. The proliferation and differentiation potentials of adipose-derived stem cells (ASCs) isolated by both methods was also investigated. METHODS: Fat grafts were collected from patients who underwent liposuction procedures by 2 different methods: traditional suction-assisted liposuction (TSAL) and vibration amplification of sound energy at resonance (VASER). One portion of the lipoaspirates was implanted into the subcutaneous layer of nu mice for 4 and 12 weeks. ASCs were isolated from the other portion of the lipoaspirate and subjected to proliferation and differentiation assays. RESULTS: Although in vivo fat grafting presented similar adipose tissue survival for the 2 different liposuction methods, more angiogenesis and less fibrosis was observed in the VASER group based on histologic evaluation. Furthermore, VASER-derived ASCs presented better quality in terms of cell differentiation capacity. CONCLUSIONS: The in vivo study confirmed better graft angiogenesis with less inflammation, apoptosis, and scar formation in the VASER group. ASCs harvested with VASER exhibited increased differentiation capacity compared with those obtained by TSAL, and represent an excellent source for fat grafting and regenerative medicine.

The effect of bone inhibitors on periosteum-guided cartilage regeneration.

The regeneration capacity of knee cartilage can be enhanced by applying periosteal grafts, but this effect varies depending on the different sources of the periosteal grafts applied for cartilage formation. Tibia periosteum can be used to enhance cartilage repair. However, long-term analysis has not been conducted. The endochondral ossification capacity of tibia periosteum during cartilage repair also needs to be investigated. In this study, both vascularized and non-vascularized tibia periosteum grafts were studied to understand the relationship between tissue perfusion of the periosteum graft and the effects on cartilage regeneration and bone formation. Furthermore, anti-ossification reagents were added to evaluate the efficacy of the prevention of bone formation along with cartilage regeneration. A critical-size cartilage defect (4 × 4 mm) was created and was covered with an autologous tibia vascularized periosteal flap or with a non-vascularized tibia periosteum patch on the knee in the rabbit model. A portion of the vascularized periosteum group was also treated with the anti-osteogenic reagents Fulvestrant and IL1ß to inhibit unwanted bone formation. Our results indicated that the vascularized periosteum significantly enhanced cartilage regeneration in the cartilage defect region in long-term treatment compared to the non-vascularized group. Furthermore, the addition of anti-osteogenic reagents to the vascularized periosteum group suppressed bone formation but also reduced the cartilage regeneration rate. Our study using vascularized autologous tissue to repair cartilage defects of the knee may lead to the modification of current treatment in regard to osteoarthritis knee repair.

Fe in biosynthesis, translocation, and signal transduction of NO: toward bioinorganic engineering of dinitrosyl iron complexes into NO-delivery scaffolds for tissue engineering.

Iron, the most abundant transition metal ion in humans, participates in the biosynthesis, translocation, signal transduction, and transformation of nitric oxide through its encapsulation in the form of heme, [Fe-S], and [Fe(NO)2] cofactors within a variety of enzymes and proteins. After the review on nitric oxide synthase (NOS) and soluble guanylate cyclase (sGC) for the biosynthesis and detection of NO, in this report, we discuss the natural utilization of the [Fe(NO)2] motif for translocation of endogenous NO and the translational development of synthetic dinitrosyl iron complexes (DNICs) for biomedical applications. A mechanistic study of NO-release and NO-transfer reactivity of structure-characterized DNICs promoted the discovery of cell-penetrating and in vivo NO-delivery reactivity for treatment of cancer and wound healing in diabetes. Beyond activation of sGC and vasodilation, phase I/II clinical trials of glutathione-bound DNICs (Oxacom®) against hypertension encourage bioinorganic engineering of DNICs into scaffolds for tissue regeneration and repair relying on anti-bacterial, anti-inflammation, cytoprotective, and proliferative effects of NO.

Critical Ischemia Time, Perfusion, and Drainage Function of Vascularized Lymph Nodes.

BACKGROUND: Vascularized lymph node transfer is a promising surgical treatment for lymphedema. This study investigated the effect of ischemia on the lymphatic drainage efficiency of vascularized lymph node flaps and the critical ischemia time of lymph nodes. METHODS: Twenty-four lymph nodes containing groin flaps in 12 Sprague-Dawley rats were dissected. Clamping of the vascular pedicle was performed for 0, 1, 3, 5, 6, or 7 hours; then, each was allowed to reperfuse by means of the vascular pedicle for 1 hour. Perfusion and ischemic changes were assessed using indocyanine green lymphography; laser Doppler flowmetry; and histologic studies with associated lymphatic vessel endothelial hyaluronan receptor-1, CD68, 4',6-diamidino-2-phenylindole, terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling, and glutathione assay stains. RESULTS: The mean latency period of the groin lymph node flaps was 247 ± 67, 83 ± 15, 72 ± 42, 30 ± 18, and 245 ± 85 seconds in the 0-, 1-, 3-, 5-, and 6-hour groups, respectively. Perfusion detected by laser Doppler was 85.2 ± 14.5, 87.2 ± 36.7, 129.8 ± 33.7, 140.4 ± 148.5, 156.1 ± 91.4, and 41.2 ± 34.8 perfusion units at ischemia times of 0, 1, 3, 5, 6, and 7 hours, respectively. Cell damage measured by glutathione was 46.8 ± 10.2, 67.7 ± 14.2, 62.8 ± 15.4, 126.6 ± 5.9, 259.0 ± 70.3, and 109.1 ± 27.5 at ischemia times of 0, 1, 3, 5, 6, and 7 hours, respectively. Histologically, as ischemia time increased, hemorrhage and congestion became more severe. CONCLUSIONS: The critical ischemia time of vascularized lymph nodes is 5 hours in the rodent animal model, verified by indocyanine green lymphatic fluid uptake, laser Doppler perfusion, and histologic assessments. Interestingly, lymphatic drainage and perfusion of vascularized lymph nodes were improved with an increased ischemia time before the critical 5 hours was reached.

Periosteal Osteogenic Capacity Depends on Tissue Source.

Periosteal osteogenic capacity can be exploited to enhance bone formation in the fields of tissue engineering and regenerative medicine. Despite this importance, there have been no studies examining the composition, structure, and osteogenic capacity of periostea from different bone sources. In this study, structure and osteogenic factor content were compared among periostea from rib, calvarial, femoral, and tibial bones, in which the native bones of these four regions were harvested and subjected to histological analysis. The osteogenic capacity of grafted periosteum was evaluated using an in vivo vascularized pedicle model of bone tissue engineering. Poly(ethylene glycol)-poly(l-lactic acid) (PEG-PLLA) copolymer hydrogels were seeded with bone marrow mesenchymal stem cells and implanted with grafted periosteum harvested from either calvarial or tibial bone, which were representative of thin and thick native periostea, respectively. The cambium layer thickness of periostea from the femoral and tibial bones (36.9% ± 2.5% and 36.8% ± 2.6%) was greater than that from the calvarial and rib bones (26.8% ± 2.4% and 25.5% ± 1.9%). The osteocalcin and alkaline phosphatase levels were comparatively higher in the femoral and tibial periostea than those in periostea harvested from the calvarial and rib bones. The construct implanted with grafted tibial periosteum resulted in greater neo-bone regeneration and higher osteocalcin and alkaline phosphatase expression. This study is the first investigation of the osteogenic capacity of periostea from diverse sources. The results can be used to guide clinical strategies that exploit periostea for tissue engineering and clinical applications.