One million hMSC-TERT (2 implants, 1 mouse) and iHBFCCD105 (4 implants, 3 mice) were mixed with 40 mg hydroxyapatite/tricalcium phosphate (HA/TCP) ceramic powder (Zimmer Scandinavia, Albertslund, Denmark), incubated at 37 °C at 5% CO2 atmosphere overnight and then implanted subcutaneously in the dorsal side of NOD.CB17-PrkdcScid/J mice (Charles River, France) [29 (link)]. Implants were removed after 8 weeks, transferred to 4% neutral buffered formalin for 24 h followed by incubation in formic acid for 3 days. The processed implants were paraffin-embedded, sectioned, and stained as described [30 (link)] with human-specific vimentin (Thermo Fisher Scientific, clone SP20) antibody or by hematoxylin-eosin [31 ].
Ha tcp ceramic powder
Manufactured by Zimmer Biomet
Sourced in Denmark, United States
HA/TCP ceramic powder is a synthetic bone graft material composed of hydroxyapatite (HA) and tricalcium phosphate (TCP). It is designed to provide a porous scaffold for bone cell growth and regeneration. The product is available in various particle sizes to accommodate different clinical applications.
Automatically generated - may contain errors
Lab products found in correlation
Cb17 prkdcscid j mice, by Charles River Laboratories (1 mentions)
Biotin labeled goat anti rabbit igg, by Vector Laboratories (1 mentions)
Nod cb17 prkdcscid j, by Charles River Laboratories (1 mentions)
Nod mrkbomtac prkdcscid female mice, by Taconic Biosciences (1 mentions)
Ha tcp, by Zimmer Biomet (1 mentions)
10 protocols using ha tcp ceramic powder
1
Subcutaneous Implantation of hMSC-TERT and iHBFC^CD105
2
Bone Regeneration with HA/TCP and BMP4
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The primary cultured mouse bone‐derived cells (1 × 106) were mixed with 100‐mg hydroxyapatite/tricalcium phosphate (HA/TCP) ceramic powder (Zimmer, Warsaw, IN, USA) alone or with BMP4 (5 μg; Peprotch, Rocky Hill, NJ, USA) in a 0.5% fibrin gel, and then transplanted s.c. into immunocompromised mice (NIH‐bg‐ nu‐xid; Harlan Laboratories, Indianapolis, IN, USA) for 6 and 12 weeks.
For histomorphometric analysis of newly formed mineralized tissue, samples were harvested and fixed in 4% PFA, decalcified in 10% EDTA (pH 7.4), embedded in paraffin, and stained with H&E, Masson's trichrome (Polysciences Inc., Warrington, PA, USA), or processed for immunohistochemistry. For immunohistochemistry, proteins were detected with anti‐BSP(17 ) at a dilution of 1:100 as the primary antibody and a biotin‐labeled goat anti‐rabbit IgG (Vector Labs) as the secondary antibody. Tartrate‐resistant acid phosphatase staining was performed. The total mineralized area among the regenerated bone‐ and marrow‐like tissue was analyzed using the LS starter program (Olympus Soft Imaging Solutions).
For histomorphometric analysis of newly formed mineralized tissue, samples were harvested and fixed in 4% PFA, decalcified in 10% EDTA (pH 7.4), embedded in paraffin, and stained with H&E, Masson's trichrome (Polysciences Inc., Warrington, PA, USA), or processed for immunohistochemistry. For immunohistochemistry, proteins were detected with anti‐BSP(
3
Bone Regeneration via BMMSCs and HA/TCP
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We subsequently implanted 4.0×106 BMMSCs mixed with 40 mg HA/TCP ceramic powder (Zimmer Inc., Warsaw, IN, USA) under the dorsal surface of 8-week-old immunocompromised mice. The transplants were harvested at 8 weeks post implantation, fixed in 4% PFA, and decalcified with 5% EDTA, then embedded in paraffin. The sections were stained with H&E. For quantification of in vivo new bone regeneration, we calculated the area of bone formation with 10 representative images from different regions of the BMMSC implants using ImageJ software (National Institutes of Health, Bethesda, MD). Each experimental group was repeated with three independent implants.
4
Subcutaneous Implantation of Human Cells in Mice
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Procedures for injection of human cells under the skin were reviewed and approved by the Danish National Animal Experiment Inspectorate (2017-15-0201-01210). One million bMSCs (7 implants, 3 mice), myCAFs (4 implants, 3 mice) and crude primary CAFs from three different donors in passage five (13 implants, 5 mice) were mixed with 40 mg hydroxyapatite/tricalcium phosphate (HA/TCP) ceramic powder (Zimmer Scandinavia, Albertslund, Denmark), incubated at 37 °C at 5% CO2 atmosphere overnight and then implanted subcutaneously in the dorsal side of immune-deficient mice (NOD.CB17-PrkdcScid/J, Charles River, France). Implants were removed after eight weeks, transferred to 4% neutral buffered formalin for 24 h followed by incubation in formic acid for 3 days. The processed implants were paraffin embedded, sectioned and stained as described [46 (link)] with human-specific vimentin (clone SP20) antibody and hematoxylin ± eosin. The primary CAFs included in this experiment were cultured under conditions specified above for breast fibroblasts as well as those described for bMSCs, and the number of implants refers to the combined total. Mice transplanted with bMSCs served as a positive control for bone formation and material from one of these experiments was included in a previous study [5 (link)].
5
Bone Formation in Subcutaneous Implants
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Cells were cultured in CIM medium and 5 × 105 cells, mixed with 40 mg hydroxyapatite/ tricalcium phosphate (HA/TCP) ceramic powder (Zimmer Scandinavia Albertslund, Denmark) and implanted subcutaneously in 2-month-old NOD/MrkBomTac-Prkdcscid female mice (Taconic, Ry, Denmark) (n = 6 implants/cell line). Implants demineralized in EDTA solution ((25% W/V), pH = 7.1), paraffin embedded, sectioned, and stained by eosin/hematoxylin. The percentage of total bone area per total implant area was quantified as described previously [18 (link)].
6
Analysis of Bone Formation from DPSC Implants
7
Scaffold Material Comparison for DPSC
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Three different scaffold materials (40 mg) were used as follows; a. PLDL in a 80/20 molar ratio with an inherent viscosity midpoint of 5.8 dl/g (Purasorb PLDL, Purac, Holland) b. PDL with an inherent viscosity midpoint of 2.0 dl/g. (Purasorb PDL, Purac, Holland) c. HA-TCP ceramic powder (Zimmer, Warsaw, Indiana, USA)
The polymer scaffolds, PLDL and PDL, were dissolved in dioxane before being mixed with the cells. The cell suspensions, containing ~1x10 6 DPSC cultured to the third passage, were mixed with the appropriate scaffold material, and transplantation was performed as described below.
The polymer scaffolds, PLDL and PDL, were dissolved in dioxane before being mixed with the cells. The cell suspensions, containing ~1x10 6 DPSC cultured to the third passage, were mixed with the appropriate scaffold material, and transplantation was performed as described below.
8
In vivo Dentinogenic Capacity Evaluation
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To analyze in vivo dentinogenic capacity, the cells were implanted subcutaneously with HA/TCP ceramic powders (40 mg, Zimmer Inc., Warsaw, IN, USA) into BALB/cAJcl-nu/nu mice as described in Supplementary Methods 49 (link)50 (link)51 (link)52 (link) (Supplementary Figure 2 ). Eight weeks after the surgery, the implants were analyzed histologically. For self-renewal assay, the cells were sequentially transplanted as described in Supplementary Methods 49 (link)50 (link) (Supplementary Figure 2 ).
9
In Vitro Calcified Tissue Formation
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In vitro calcified tissue formation was performed according to the previous studies [1 (link), 25 (link)]. Briefly, SCAP (P3, 4 × 106), which were cultured under the growth condition, were mixed with hydroxyapatite/tricalcium phosphate (HA/TCP) ceramic powders (40 mg, Zimmer Inc., Warsaw, IN). The mixture was implanted subcutaneously into the dorsal surface of Balb/cAJcl-nu/nu immunocompromised mice (male, 8-week-old; CLEA Japan, Tokyo, Japan). As the control, HA/TCP (40 mg, Zimmer Inc.) alone without SCAP was implanted. Eight weeks after the transplantation, the implants were harvested for histological assays. Some transplants were used for RT-qPCR analysis.
10
Subcutaneous Bone Tissue Engineering
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Approximately 4.0 × 106 BMMSCs were mixed with HA/TCP ceramic powders (40 mg; Zimmer Inc.) and subcutaneously implanted into 8-wk-old immunocompromised mice (Gronthos et al., 2000 (link)). At 8 wk after implantation, the transplants were harvested, fixed in 4% PFA, and decalcified with 5% EDTA, pH 7.4, followed by paraffin embedding. The 6-µm paraffin sections were stained with H&E chemical staining. Total BV/TV was quantified by ImageJ software (National Institutes of Health).
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