Does the Embryologic Limb Rotation Modify the Distribution of Dermal Collagen and Elastic Fibers in Different Planes of Human Extremities?

Naveen Kumar¹, Pramod Kumar², Satheesha Nayak B³, Melissa Glenda Lewis⁴,

Ashwini Aithal P³, Ravi Bhaskar⁵

¹Department of Anatomy, Ras Al Khaimah College of Medical Sciences, RAK Medical and Health Sciences University, Ras Al Khaimah, UAE (Present).Former Faculty of Melaka Manipal Medical College,

Manipal Academy of Higher Education, Manipal, India.

²Consultant Plastic Surgeon, King Fahad Central Hospital, Jazan, Saudi Arabia.

³Division of Anatomy, Department of Basic Medical Sciences,

Manipal Academy of Higher Education, Manipal, India.

⁴Indian Institute of Public Health, Shillong, Meghalaya, India.

⁵Department of Anatomy, Sapthagiri Institute of Medical Sciences and Research Centre, Bengaluru, India.

*Corresponding Author E-mail: ashwini.anat@gmail.com, ashwini.aithal@manipal.edu

ABSTRACT:

Background: The collagen and the elastic fiber content of the dermis is known to show the asymmetric distribution across the planes of the body region. During the period of limb development, the upper and lower limbs rotate 90 degrees laterally and medially respectively. Objectives: The study aims to check if the limb bud rotation during embryonic development favors the unequal distribution of the dermal connective tissue fiber content distribution. Methods: A total of 240 skin samples from the forearm and thigh regions of adult human cadavers were collected in horizontal and vertical directions from the anatomical and oblique planes. The tissues were processed with tissue-quant analysis. The data were analyzed statistically using SPSS. Results: There were no significant differences in the pattern of distribution of the collagen and elastic fiber contents in the tissues of horizontal and vertical directions from the two planes of forearm and thigh regions. However significant Spearman's correlation was observed in both elements in the thigh region but only the collagen content in the forearm region. Conclusion: The pattern of distribution of connective tissue fibers in the dermis is regulated by the mechanical communications between the cells and their extracellular matrices. Hence, the significant role of embryologic limb rotation in the event of unequal distribution of dermal collagen and elastic fibers in the limb region is to be ruled out.

KEYWORDS: Limb rotation, Anatomical plane, Oblique plane, Extracellular matrix.

INTRODUCTION:

Embryological development of the limbs occurs by the beginning of the 5^th week of intrauterine life with the appearance of forelimb and hind limb buds. During the 7^th week of gestation, the upper limb rotates 90 degrees laterally and the lower limb rotates 90 degrees medially.¹While the epidermis including hair and nails are derived from the ectoderm, the dermis is contributed by the dermatome component of the somites.

At birth, collagen fiber bundles in the papillary dermis exist in high density until adulthood. But, in the reticular dermis, it is thought to get diminished gradually.²After birth, the remodeling process of the collagen may be one of the causes of the decreased synthesis even loss of density of its bundles. The fetal dermis, with essential higher expression of type I and III procollagens, undergo subsequent increase during postnatal life thereafter exhibit decline as the skin progress.³ The collagen and elastic fiber content in the dermis have no disparity concerning gender and age.^4,5 However, the asymmetric distribution of both collagen and elastic fibers throughout the body has been well documented across horizontal and vertical planes of the human body.^6-8Further probe into this, the current histomorphometric study was undertaken to find out any effect of embryological limb rotation results in the impaired distribution of dermal collagen and elastic fibers in the human limb region. For this, the skin samples were obtained in horizontal and vertical directions from two different planes, the anatomical plane, and the plane oblique to it from forearm and thigh regions.

MATERIALS AND METHOD:

Study population:

Two hundred and forty skin samples of materials collected from thirty formalin embalmed healthy-looking adult human cadavers with the age ranging approximately between 60 and 70 years. The flexor compartment of the forearm from the upper limb and the adductor compartment of the thigh from the lower limb were chosen for this study. From each of these regions, four elliptical skin samples with a diameter of 1.0cm long x 0.5cm wide were taken and processed histologically. The topographic specification of the sample collection is described below (Figure 1).

a. Forearm: Skin tissues were collected at the middle of the anterior compartment of the forearm.in horizontal (H₁) and vertical (V₁) directions along the anatomical plane and in the directions of horizontal (H₂) and vertical (V₂) along the oblique plane.

b. Thigh: Skin tissues were collected in the middle of the adductor compartment of the thigh in horizontal (H₁) and vertical (V₁) directions along the anatomical plane and in the directions of horizontal (H₂) and vertical (V₂) along the oblique plane.

Here, the skin samples taken in the anatomical plane represent the post-rotation of the limbs and those in the oblique plane represent the pre-rotation of the limbs.

Tissue processing:

The skin tissues were fixed in a 10% formaldehyde solution and processed with the paraffin impregnation. Histological sections with 5μm thickness were obtained from the rotary microtome and stained the sections with a Verhoeff-Van Gieson (VVG) method of the special stain as per standard protocol.⁹ The selective staining of the collagen and elastic fibers are made possible with this technique. From each VVG stained section, a minimum of three photomicrographs was obtained under 20x magnification using an inverted phase-contrast microscope attached with ProgRes CapturePro 2.1, Jenoptik microscopic camera. These images were taken with the resolution of 694× 516 VGA for the image analysis by the "tissue-quant" method. The images were captured from the dermis area just beneath the epidermis layer, with the involvement of papillary and reticular layers.

Image analysis:

The quantitative fraction of the collagen and elastic fiber content distribution was analyzed by image analysis technique using the software Tissue Quant (Version 1.0). This software works on the principle of a number of pixels occupied by the shades of the desired color (Red for Collagen and black for elastic in VVG stained sections) to be analyzed. The total number of pixels corresponding to the color of interest is subsequently accounted to the percentage by the appropriate calculation.

Statistical analysis:

The Paired t-test was performed to determine whether there exists a difference in collagen and elastic fiber contents in the dermis of skin tissues taken along two different (anatomical and oblique) planes at forearm and thigh regions. Mean with standard deviations are reported as the difference follows the mostly normal distribution. The 𝑃<0.05 is considered to be statistically significant. The statistical analysis was performed with the software SPSS 17.0 for Windows (SPSS Inc., Chicago, IL, USA).

Spearman rank correlation coefficient with bootstrap confidence intervals (replication =1000) was reported to assess the correlation of the collagen and elastic contents in assessed directions of the forearm and thigh regions.

RESULTS:

The mean content of collagen between horizontal anatomical (CH1) and horizontal oblique (CH2), and between vertical anatomical (CV1) and vertical oblique (CV2) directions at forearm and thigh regions have shown no significant differences in the distribution pattern. Similarly, the mean elastic fiber content between horizontal anatomical (EH1) and horizontal oblique (EH2) and between vertical anatomical (EV1) and vertical oblique (EV2) directions at both the regions also did not show statistical differences in the pattern of distribution (Table-1).

The Spearman's correlation coefficients between the above-mentioned variables are tabulated in table 2. A positive significance (p<0.05) with moderate correlation was observed in both the collagen and elastic distribution only at the thigh region. In the forearm, the coefficients were statistically significant (p<0.05) for collagen but not for elastic fibers (Table 2). The scatter plot diagrams with the regression line are depicted in figures 2-5.

Figure 1: Topographic location of skin tissue collection from the forearm (a) and thigh (b) regions of limbs. H₁ and V₁- horizontal and vertical sections in the anatomical plane. H₂ and V₂ – horizontal and vertical sections in oblique directions

Table 1. Quantitative fraction analysis of collagen (C) and Elastic (E) fibers in the skin tissues obtained from two different (horizontal and vertical) directions of anatomical and oblique planes of forearm and thigh regions

Characteristics	Mean± SD	P-value
Forearm- CH1	52.98±12.62	0.45
Forearm-CH2	51.08±11.91	0.45
Forearm- CV1	52.34±12.49	0.52
Forearm-CV2	50.64±15.57	0.52
Forearm-EH1	14.28±04.89	0.63
Forearm-EH2	13.64±06.66	0.63
Forearm-EV1	14.22±06.68	0.40
Forearm-EV2	15.60±07.46	0.40
Thigh- CH1	52.58±12.53	0.83
Thigh- CH2	52.15±12.68	0.83
Thigh- CV1	52.99±13.63	0.94
Thigh- CV2	53.17±10.47	0.94
Thigh-EH1	14.93±6.96	0.97
Thigh-EH2	14.98±8.73	0.97
Thigh-EV1	12.45±5.55	0.84
Thigh-EV2	12.69±6.03	0.84
SD: standard deviation

C- Collagen

E- Elastic

H₁- Horizontal direction in the anatomical plane

H₂- Horizontal direction in oblique plane

V₁- Vertical direction in the anatomical plane

V₂- Vertical direction in oblique plane

Table 2: Spearman’s Correlation (Rho) coefficient comparison of collagen and elastic fibers distribution in two directions concerning anatomical and oblique planes at forearm and thigh regions

Characteristics	Spearman’s Rho Correlation (95% CI)	P-value
Forearm- CH1	0.42(0.09,0.74)	0.012^*
Forearm-CH2
Forearm- CV1	0.51(0.27,0.75)	<0.001^*
Forearm-CV2
Forearm-EH1	0.27(-0.14,0.67)	0.20
Forearm-EH2
Forearm-EV1	0.13(-0.26,0.52)	0.51
Forearm-EV2
Thigh- CH1	0.59(0.31,0.87)	<0.001^*
Thigh- CH2
Thigh- CV1	0.44(0.15,0.74)	0.003^*
Thigh- CV2
Thigh-EH1	0.61(0.38,0.85)	<0.001^*
Thigh-EH2
Thigh-EV1	0.40(0.10,0.69)	0.009^*
Thigh-EV2
^*Statistically significant at 5% level of significance Bootstrap confidence intervals (CI) replication =1000

C- Collagen

E- Elastic

H₁- Horizontal direction in the anatomical plane

H₂- Horizontal direction in the oblique plane

V₁- Vertical direction in the anatomical plane

V₂- Vertical direction in the oblique plane

DISCUSSION:

In the present study, histo-morphometric evaluation of dermal collagen and elastic fibers from non-stretchable areas of upper and lower limbs were evaluated. In both the regions, no significant changes in the distribution of these connective tissue fibers between two planes of the region were noted. That means, the embryological limb rotation is not affecting the significant changes in the distribution of dermal connective tissue fibers.

The minimal effect of movement at these areas, contrary to stretchable joint areas is the main fact that could be justified for the non-disparity of dermal connective tissue elements despite limb rotation in early embryogenesis. Collagen content in the dermis is often in higher distinct at different directions owing to repeated stress in the specific region as a result of associated elastic fiber content or physical stretching.^10,11

An experimental study showed when the limbs are shifted surgically from the lumbosacral to the thoracic region, the muscles may have composite origin but the connective tissues in the skin are restricted to their level of origin. This pattern was observed in the mid-thigh region of shifted limbs.¹²This is the major concept in which there is no effect of limb rotation in the unequal distribution of dermal collagen and elastic fibers across anatomical and oblique planes.

Cao et all proposed that the extracellular matrix of the collagen fiber bundle directs the fibroblast migration as an extracellular cue.¹³ Besides, cell migration because of a cellular response increases the collagen bundle alignment as evidence in fibrotic skin. Hence, higher accumulation and morphological changes in the dermal collagen is a pathological feature in which loss of normal morphology is significantly altered. ^14,15,16A similar manifestation as a probable result of underlying tissue remodelling after the injury is also observed in burn wound skin and physiological aging of the skin. ^17,18,19

The significant positive correlation between the dermal collagen and elastic fibers content distribution across two different planes of the thigh region is interesting findings of this study when compared to only the significant correlation seen for collagen but not for elastic fibers in the forearm region. The duration of exposure and frequent pronation and supination activities of the forearm in comparison with rotation actions of the thigh is a possible case that could be postulated.

As the mechanical communications between the cells and their extracellular matrix regulate the growth and migration of the connective tissue fibers, we conclude that embryonic limb rotation has no role in their asymmetric distribution at any plane of the limbs.

LIMITATIONS OF THE STUDY:

The comparison of dermal connective tissue content in the representative regions of the upper and lower limb was not made as done here among the distal part of the upper limb with the proximal part of the lower limb.

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Received on 18.07.2022 Modified on 07.10.2022

Research J. Pharm. and Tech 2023; 16(10):4525-4529.

DOI: 10.52711/0974-360X.2023.00737