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. 2024 Apr 26;24(1):23.
doi: 10.1186/s12896-024-00847-4.

Multiprotein collagen/keratin hydrogel promoted myogenesis and angiogenesis of injured skeletal muscles in a mouse model

Atieh Rezaei Namjoo  1 , Ayla Hassani  2   3 , Hassan Amini  4 , Fateme Nazaryabrbekoh  1 , Sepideh Saghati  5 , Mohammad Ali Ebrahimi Saadatlou #  6 , Ali Baradar Khoshfetrat  2 , Nafiseh Didar Khosrowshahi  2 , Reza Rahbarghazi #  7   8
Affiliations

Multiprotein collagen/keratin hydrogel promoted myogenesis and angiogenesis of injured skeletal muscles in a mouse model

Atieh Rezaei Namjoo et al. BMC Biotechnol. .

Abstract

Volumetric loss is one of the challenging issues in muscle tissue structure that causes functio laesa. Tissue engineering of muscle tissue using suitable hydrogels is an alternative to restoring the physiological properties of the injured area. Here, myogenic properties of type I collagen (0.5%) and keratin (0.5%) were investigated in a mouse model of biceps femoris injury. Using FTIR, gelation time, and rheological analysis, the physicochemical properties of the collagen (Col)/Keratin scaffold were analyzed. Mouse C2C12 myoblast-laden Col/Keratin hydrogels were injected into the injury site and histological examination plus western blotting were performed to measure myogenic potential after 15 days. FTIR indicated an appropriate interaction between keratin and collagen. The blend of Col/Keratin delayed gelation time when compared to the collagen alone group. Rheological analysis revealed decreased stiffening in blended Col/Keratin hydrogel which is favorable for the extrudability of the hydrogel. Transplantation of C2C12 myoblast-laden Col/Keratin hydrogel to injured muscle tissues led to the formation of newly generated myofibers compared to cell-free hydrogel and collagen groups (p < 0.05). In the C2C12 myoblast-laden Col/Keratin group, a low number of CD31+ cells with minimum inflammatory cells was evident. Western blotting indicated the promotion of MyoD in mice that received cell-laden Col/Keratin hydrogel compared to the other groups (p < 0.05). Despite the increase of the myosin cell-laden Col/Keratin hydrogel group, no significant differences were obtained related to other groups (p > 0.05). The blend of Col/Keratin loaded with myoblasts provides a suitable myogenic platform for the alleviation of injured muscle tissue.

Keywords: Collagen/keratin hydrogel; Mouse; Myogenic properties; Tissue engineering; Volumetric loss.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Surgical procedure for the induction of volumetric muscle loss in a mouse model after biceps femoris muscle injury and transplantation of myoblast-laden Col/Keratin hydrogel. After shaving and disinfection, a surgical incision was made to get access to the biceps femoris muscle mass. For volumetric muscle loss, segments of skeletal muscle surgical were removed surgically. The process followed by the injection of Col, and Col/Keratin hydrogels with or without the myoblasts. After being solidified, the incision line was carefully sutured and the animals were kept for 15 days
Fig. 2
Fig. 2
FTIR analysis of Col, Keratin, and Col/Keratin hydrogels (A). Data confirmed that the mixture of Col and Keratin yielded prominent peak shifting in the amide I area from 3442 cm− 1 to 3418 cm− 1, amide II area from 1550 cm− 1 to 1575 cm− 1, C-H bending vibration area from 1454 cm− 1 to 1467 cm− 1, and the emergence of C-H symmetric stretching vibration at 2851 cm− 1. Monitoring gelation time in Col (0.5% w/v)/(0.5% w/v)Keratin hydrogel compared to the (0.5% w/v) Col hydrogel alone (B). The addition of Keratin to Col solution prologned the gelation time from 40 to 50 min
Fig. 3
Fig. 3
Rheological analysis of Col and Col/Keratin hydrogels. Data indicated Col hydrogel alone exhibited a higher viscosity and stiffness rate compared to the Col/Keratin group
Fig. 4
Fig. 4
H & E staining of injured muscle tissue after transplantation of hydrogel. Mice were allocated into Col, Col + Cells, Col/Keratin, and Col/Keratin + Cells groups. Bright-field imaging indicated that in the group that received Col hydrogel alone, numerous inflammatory cells were recruited to the injury site (black arrows), leading to the digestion and scavenging of hydrogel remnants. These features were less in other groups that received the combination of cells with hydrogel. In Col + Cells, inflammatory cells (black arrows) are at the periphery of newly generated myofibers (blue arrows). The myofibers are not aligned in a regular pattern. In the Col + keratin group, the intensity of immune cells was reduced and newly generated myofibers exhibited a more aligned pattern (blue arrows). Maximum myofiber density was achieved in the group with the transplantation of Col/Keratin and Cells. The area was filled with aligned newly generated myofibers (blue arrows) and few immune cells can be evident in intermyocyte space (black arrows). Data analysis revealed the maximum myofiber formation in the Col/Keratin + Cells group compared to the other groups (10 high-power fields). One-way ANOVA analysis with Tukey test. **p < 0.01; ***p < 0.001; and ****p < 0.0001
Fig. 5
Fig. 5
IHC staining of CD31 positive cells in different groups after transplantation of hydrogel (A). CD31+ cells are indicated using black arrows. In the control group, CD31+ cells with flattened morphology are juxtaposed with normal myocytes in the muscular tissue. In the Col group, numerous CD31+ cells can be detected within the injection site without a regular pattern, indicating the promotion of angiogenesis due to immunological response. Data indicated more aligned CD31+ cells in the Col + Cells group. The intensity and number of CD31+ cells were reduced in Col/Keratin and Col/Keratin + Cells groups. CD31+ cells can be detected at the periphery of newly generated myofiber in the Col/Keratin + Cells group. Westernblotting analysis of MyoD and myosin (B; n = 3). Data revealed the induction of MyoD in Col + Cells and Col/kertain + Cells groups, indicating the maturation of muscle progenitor cells. Despite the increase of myosin in the Col/Kertain + Cells group, the differences were statistically non-significant. One-way ANOVA analysis with Tukey test. *p < 0.05; ***p < 0.001; and ****p < 0.0001
Fig. 6
Fig. 6
Interaction of Col with keratin after physical blending with possible mechanisms
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