First immunohistochemical evidence of human tendon repair following stem cell injection: A case report and review of literature

doi:10.4252/wjsc.v13.i7.944

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Jul 26, 2021 (publication date) through Aug 25, 2024

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World Journal of Stem Cells

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1948-0210

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Baishideng Publishing Group Inc, 7041 Koll Center Parkway, Suite 160, Pleasanton, CA 94566, USA

Case Report

World J Stem Cells. Jul 26, 2021; 13(7): 944-970
Published online Jul 26, 2021. doi: 10.4252/wjsc.v13.i7.944

Table 1 Characteristics of the antibodies used in this study

Aggrecan
Immunoglobulin isotype/clone status	IgG1/mouse, monoclonal
Catalog no./provider	12/21/1-C-6/DSHB¹
Demasking of antigen	Protease XIV and Chondroitinase AC
Blocking	Bloxall SP-6000², NHS S-2000², 1:20
Dilution and incubation parameters	1:5, room temperature, 4 h
Secondary antibody used	HaM IgG BA-2000², 1:200
CD34
Immunoglobuline isotype/clone status	IgG1/mouse, monoclonal
Catalog no./provider	QBEnd-10/Thermo Scientific (Waltham, MA, United States)
Demasking of antigen	N/A
Blocking	3% H₂O₂ in methanol, NGS S-1000² 5%
Dilution and incubation parameters	1:900, 4 °C, overnight
Secondary antibody used	GaM IgG BA-9200², 1:200, in 2% BSA/PBS
CD68
Immunoglobuline isotype/clone status	KP1- IgG1 kappa/mouse, monoclonal
Catalog no./provider	M0814/Dako (Glostrup, Denmark)
Demasking of antigen	Boiling in citrate buffer (pH 6)
Blocking	Bloxall SP-6000², NHS S-2000², 1:20
Dilution and incubation parameters	1:100, 4 °C, overnight
Secondary antibody used	HaM IgG BA-2000², 1:200
Ki-67
Immunoglobuline isotype/clone status	MIB-1 IgG1 kappa/mouse, monoclonal
Catalog no./provider	M7240/Dako (Glostrup, Denmark)
Demasking of antigen	Boiling in citrate buffer (pH 6)
Blocking	Bloxall SP-6000², NHS S-2000², 1:20
Dilution and incubation parameters	1:75, 4 °C, overnight
Secondary antibody used	HaM IgG BA-2000², 1:200
Laminin
Immunoglobuline isotype/clone status	IgG2a/mouse, monoclonal
Catalog no./provider	2E8/DSHB¹
Demasking of antigen	N/A
Blocking	3% H₂O₂in methanol, NHS S-2000², 1:20
Dilution and incubation parameters	1:5, room temperature, 30 min
Secondary antibody used	HaM IgG BA-2000², 1:200
MMP-2
Immunoglobuline isotype/clone status	IgG/rabbit, polyclonal
Catalog no./provider	97779/Abcam (Cambridge, MA)
Demasking of antigen	N/A
Blocking	Bloxall SP-6000², NGS S-1000², 1:20
Dilution and incubation parameters	1:100, 4 °C, overnight
Secondary antibody used	GaR IgG BA-1000², 1:200
MMP-9
Immunoglobuline isotype/clone status	IgG/rabbit, polyclonal
Catalog no./provider	38898/Abcam
Provider	Abcam
Demasking of antigen	N/A
Blocking	Bloxall SP-6000², NGS S-1000², 1:20
Dilution and incubation parameters	1:100, 4 °C, overnight
Secondary antibody used	GaR IgG BA-1000², 1:200
Procollagen 1
Immunoglobuline isotype/clone status	IgG1/mouse, monoclonal
Catalog no./provider	SP1.D8/DSHB¹
Demasking of antigen	Boiling in citrate buffer (pH 6)
Blocking	Bloxall SP-6000², NHS S-2012², 2.5%
Dilution and incubation parameters	1:10, 4 °C, overnight
Secondary antibody used	HaM IgG BA-2000², 1:200
Tenomodulin
Immunoglobuline isotype/clone status	IgG/rabbit, polyclonal
Catalog no./provider	203676/Abcam
Demasking of antigen	N/A
Blocking	3%H₂O₂ in Methanol, NHS S-2012², 2.5%
Dilution and incubation parameters	1:500, room temperature, 30 min
Secondary antibody used	HaR IgG BA-1100², 1:200
Type I collagen
Immunoglobuline isotype/clone status	IgG1/mouse, monoclonal
Catalog no./provider	C2456/Sigma-Aldrich (St. Louis, MO, United States)
Demasking of antigen	Protease XIV and Hyaluronidase
Blocking	Bloxall SP-6000², NHS S-2000², 1:20
Dilution and incubation parameters	1:2000, room temperature, 30 min
Secondary antibody used	HaM IgG BA-2000², 1:200
Type II collagen
Immunoglobuline isotype/clone status	MIgG2A kappa light chain/mouse, monoclonal
Catalog no./provider	CIIC1/DSHB^a
Demasking of antigen	Protease XIV and Hyaluronidase
Blocking	Bloxall SP-6000², NHS S-2000², 1:20
Dilution and incubation parameters	1:6, room temperature, 30 min
Secondary antibody used	HaM IgG BA-2000², 1:200
Type III collagen
Immunoglobuline isotype/clone status	FH-7AIgG1/mouse, monoclonal
Catalog no./provider	C 7805/Sigma-Aldrich (St. Louis, MO, United States)
Demasking of antigen	ProteaseXIV and Hyaluronidase
Blocking
Dilution and incubation parameters	1:4000, room temperature, 30 min
Secondary antibody used	HaM IgG BA-2000², 1:200
Type IV collagen
Immunoglobuline isotype/clone status	MIgG1 kappa light chain/mouse, monoclonal
Catalog no./provider	M3F7/DSHB^a
Demasking of antigen	N/A
Blocking	Bloxall SP-6000²
Dilution and incubation parameters	1:5, room temperature, 30 min
Secondary antibody used	HaM IgG BA-2000², 1:200

¹The antibodies 12/21/1-C-6 (developed by Dr. Caterson B), 2E8 (developed by Dr. Engvall ES), CIIC1 (developed by Drs Holmdahl R and Rubin K) and SP1.D8 and M3F7 (developed by Dr. Furthmayr H) were obtained from the Developmental Studies Hybridoma Bank (DSHB), created by the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health of the United States, and maintained at The University of Iowa, Department of Biology, Iowa City, IA, United States.

²Provider: Vector Laboratories (Burlingame, CA, United States). BSA: Bovine serum albumin; GaM: Goat anti-mouse; GaR: Goat anti-rabbit; HaM: Horse anti-mouse; HaR: Horse anti-rabbit; IgG: Immunoglobulin G; N/A: Not applicable; NHS: Normal horse serum; NGS: Normal goat serum; PBS: Phosphate-buffered saline.

Table 2 Histological and immunohistochemical features of different regions within the second part of the biopsy that was investigated in this study

Feature	Region in Figure 4						Figure
	B	C	D	E	F	G
Presence of microvessels	++	-	+++	+/-	-	-	4
Dense, unorganized cluster of cells and microvessels	-	-	+++	-	-	-	4
Elongated cells in a chain-like arrangement	-	-	-	++	+	+++	4
Immunohistochemical detection of
CD34 in capillary endothelial cells	-	-	++	-	+++	+	5
Type IV collagen in the basement membrane of microvessels	+	-	+++	+++	+	+	6
Ki-67 in cells inside microvessel walls	+/-	-	+++	-	-	-	7
Ki-67 in cells outside microvessel walls	-	+/-	+++	-	+++	-	7
Intracellular tenomodulin	+	-	+++	+/-	-	-	8
Extracellular tenomodulin	-	-	+++	-	-	-	8
Type I procollagen	-	+/-	+++	+++	+++	+	9
Unorganized type I collagen	+/-	-	+++	+++	-	-	10
Organized, slightly undulating Type I collagen	-	-	-	+/-	++	+++	10
Type III collagen	+	+/-	+	+	-	-	11
Intracellular laminin	+/-	+/-	+++	+++	+	+	12
Extracellular laminin	-	-	+++	+	-	-	12
Intracellular MMP-2	+/-	+/-	+++	++	++	+	13
Extracellular MMP-2	-	-	+++	++	+++	+	13
Intracellular MMP-9	+/-	+/-	+/-	-	+/-	+/-	14
Extracellular MMP-9	-	-	-	-	-	-	14
CD68	+/-	+/-	++	+	+/-	+/-	15

Absence or presence of individual features is coded as – (absence), +/- (minimal presence), + (slight presence), ++ (significant presence) and +++ (overwhelming presence). Column "Figure" indicates the corresponding figures in which detailed documentation is provided. MMP: Matrix metalloproteinase. CD: Cluster of differentiation; MMP: Matrix metalloproteinase.

Table 3 Bonar scores of all regions B-G in Figures 4-15 of the second part of the investigated biopsy

	Azan staining						CD34						Type IV collagen
	B	C	D	E	F	G	B	C	D	E	F	G	B	C	D	E	F	G
Cell morphology	3	3	3	2	2	2	2	2	3	2	3	2	3	3	3	1	2	1
Collagen arrangement	3	3	3	1	2	1	-	-	-	-	-	-	-	-	-	-	-	-
Cellularity	1	3	1	1	1	1	3	3	2	2	1	1	1	2	2	1	1	2
Vascularity	3	2	3	1	2	2	0	0	3	1	3	0	2	2	2	2	1	1
Ground substance	0	0	0	0	0	0	1	1	0	0	0	0	0	0	0	0	0	0
	Ki-67						Tenomodulin						Type I procollagen
	B	C	D	E	F	G	B	C	D	E	F	G	B	C	D	E	F	G
Cell morphology	3	3	3	2	1	1	3	3	3	2	2	1	3	3	2	2	2	1
Collagen arrangement	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Cellularity	3	3	1	2	2	2	3	3	2	1	1	1	3	3	2	2	1	2
Vascularity	2	1	3	1	0	0	2	2	3	1	1	1	1	1	3	1	1	1
Ground substance	1	1	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0	0
	Type I collagen						Type III collagen						Laminin
	B	C	D	E	F	G	B	C	D	E	F	G	B	C	D	E	F	G
Cell morphology	3	3	3	2	1	1	3	2	3	2	1	1	3	3	3	2	2	2
Collagen arrangement	3	3	2	2	1	2	-	-	-	-	-	-	-	-	-	-	-	-
Cellularity	3	3	1	1	2	1	1	2	1	1	1	1	3	3	2	2	1	1
Vascularity	1	1	2	1	1	1	3	2	3	2	1	1	1	1	3	1	1	1
Ground substance	0	0	0	0	0	0	0	0	0	0	0	0	1	1	0	0	0	0
	MMP-2						MMP-9						CD68
	B	C	D	E	F	G	B	C	D	E	F	G	B	C	D	E	F	G
Cell morphology	3	3	3	2	2	2	3	2	3	3	2	2	3	2	2	2	1	1
Collagen arrangement	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-	-
Cellularity	2	3	2	2	1	1	3	3	2	2	1	1	3	3	1	2	1	1
Vascularity	2	1	2	1	1	2	1	1	3	1	1	2	2	1	3	2	1	1
Ground substance	1	1	0	0	0	0	1	1	0	0	0	0	1	1	0	0	0	0

Note that collagen arrangement was only assessed on sections that showed type I collagen (Azan staining, type I collagen). CD: Cluster of differentiation; MMP: Matrix metalloproteinase.

Table 4 Number of cells per 45000 μm² area each in all regions B-G in Figures 4-15 of the second part of the investigated biopsy

Region	B	C	D	E	F	G
Azan staining	64	9	291	150	358	163
CD34	0	0	31	0	58	0
Type IV collagen	11	35	9	8	1	2
Ki-67	10	9	54	0	39	0
Tenomodulin	18	0	20	8	0	0
Type I procollagen	0	3	38	42	109	12
Type I collagen	5	5	121	116	82	78
Type III collagen	33	2	42	7	1	0
Laminin	8	4	30	50	11	13
MMP2	15	6	29	21	21	12
MMP9	3	3	22	0	12	14
CD68	4	3	28	14	4	3

Note that in case of Azan trichrome stain and immunohistochemical detection of type I collagen, type III collagen and type IV collagen all cells were counted, whereas in case of cluster of differentiation (CD)34, CD68, Ki-67, laminin, matrix metalloproteinase (MMP)-2, MMP-9, tenomodulin and type I procollagen only immunopositive cells were counted.

Citation: Alt E, Rothoerl R, Hoppert M, Frank HG, Wuerfel T, Alt C, Schmitz C. First immunohistochemical evidence of human tendon repair following stem cell injection: A case report and review of literature. World J Stem Cells 2021; 13(7): 944-970
URL: https://www.wjgnet.com/1948-0210/full/v13/i7/944.htm
DOI: https://dx.doi.org/10.4252/wjsc.v13.i7.944