The development of the tensor vastus intermedius during the human embryonic period and its clinical implications

Abstract

The tensor vastus intermedius (TVI) is a newly discovered muscle located in the anterolateral thigh area and is considered the fifth component of the quadriceps femoris muscle. There have been several papers describing its anatomical and morphological features in detail; however, many features of this muscle, such as its ontology or kinetic functions, remain unknown. The purpose of this study was to determine the initial appearance of the TVI muscle in human embryonic development and to investigate its growth and development. Histological observations were performed on 30 lower limbs of 15 human embryos from Carnegie stage (CS) 21, 22, and 23 (with crown‐rump length ranging from 18.7 to 28.7 mm). Myocyte clusters of the TVI were observed between the vastus lateralis and intermedius muscles in 7 out of 10 limbs in CS 22, indicating that the TVI arises during this stage. In CS 23, the TVI was clearly present in all specimens except one. However, neither the aponeurosis nor the tendonous structure of the TVI were observed in these embryonic stages. Formation of the conventional four components of the quadriceps muscle is completed within CS 21; therefore, our results suggest that the TVI is the last element to develop in the quadriceps femoris complex. It is posited that after the embryonic period, the TVI continues to grow, while forming the tendinous structure toward the patella and receiving vascular supply from certain vascular branches. The clinical significance of these findings is that orthopedists and plastic surgeons who perform surgical procedures within the anterolateral thigh (ALT) area should be aware of the anatomy and development of the TVI in order to reduce surgical complications. Our present research aims to contribute to a deeper understanding of the morphogenesis of the TVI and the other femoral extensor muscles.

The tensor vastus intermedius (TVI) is recently considered as the fifth element of the quadriceps femoris, which has been discovered as a thin muscle with a proximal muscle lamella combining a distal aponeurotic structure. In the present study using the serial sections of the human embryos, the TVI was histologically observed between the vastus lateralis (VL) and vastus intermedius(VI), and was first appeared in Carnegie Stage 22. We classified the embryonic TVI into two groups according to its original muscle, the VL or VI, which suggests the differentiation of morphological variations of the adult TVI has already directed during embryonic period.

1. INTRODUCTION

The tensor vastus intermedius (TVI) is the fifth element of the quadriceps muscle group, first reported in 2016 by Grob, Ackland, et al. (2016). Previously, this element had likely been perceived as a morphological variation of the quadriceps tendon structure around the knee, as this is how it was described in previous studies (Zeiss et al., 1992; Waligora et al., 2009; Grob et al., 2017). Currently, the TVI is generally accepted and defined among researchers as a separate muscle and studies continue to investigate its precise morphology (Grob, Manestar, et al., 2016b, 2017; Grob et al., 2017; Rajasekaran & Hall, 2016).

The TVI exists between the vastus lateralis (VL) and intermedius (VI) muscles and under the rectus femoralis (RF), forming a muscle belly in the proximal thigh and continuing distally to a thin and broad aponeurosis and tendinous structure (Grob, Ackland, et al., 2016). The TVI originates from the anterolateral surface of the greater trochanter and inserts into the medial facet of the patella. Even though the muscle belly of the TVI can be clearly separated from that of the VI and VL, in some cases, it becomes conjoined with either or both of these two adjacent muscles as it progresses distally. Grob, Ackland, et al. (2016) classified the morphologic variations of the TVI into four different subtypes with respect to aponeurotic fusions with the VL and VI: Independent‐type, VL‐type, VI‐type, and Common‐type. In the aforementioned study, Independent‐type was the most common, observed in a little less than half of the specimens, and was defined by a TVI tendon that can be easily separated from both the VL and VI. When the aponeurosis of the TVI is easily separated from the VI but not the VL, it is referred to as VL‐type; when it expresses the opposite pattern, it is referred to as VI‐type. Common‐type was an unusual variant in the study by Grob et al., and is defined by a TVI that shares its origin with the VL and VI between the intertrochanteric line and the greater trochanter and represents a common and inseparable aponeurosis in the distal site. In addition to these four types, another morphologic anomaly where the TVI had two heads and two muscle bellies was observed. Following the work of Grob et al. in a Swiss population, Veeramani and Gnanasekaran (2017) reported quantitative measurements and statistical distribution of these types of the TVI using 36 dissected limbs of South Indian adult cadavers. The sample group in their study had a different ratio of morphologic variations, with the most common being Independent‐type, followed, in order, by VL‐type, Common‐type, and VI‐type. While these two reports above focused on the variation of tendinous structure, Cariello et al. (2019) from Vassouras University in Brazil, described the form of the TVI belly, reporting a case of a bilateral presentation of the TVI with bulky muscle bellies (Cariello et al. 2019). These three reports from different countries and regions give us a realization that the TVI is present in diverse ethnic groups; however, no literature on the prevalence of the TVI in an Asian population currently exists at this time.

The quadriceps femoris muscle generally functions as the extensor apparatus of the leg and contributes to patellar stability. Four individual muscles and tendons interact with each other to create kinetic motions at the knee joint (Andrikoula et al., 2006). The specific function of the TVI as a component of the quadriceps complex is unclear. Grob et al. speculated that, from a morphological point of view, the TVI plays a role in controlling the motion of the patella and medializing the action of the VI by exerting tension on VI aponeurosis, considering the TVI as a type of “tensor of the VI” (Grob, Ackland, et al., 2016).

From a clinical perspective, this additional muscle may rarely cause any serious disabilities in most forms of treatments or rehabilitative management, but some orthopedic or reconstructive operations within the femoral area may require an updated anatomical knowledge (Waligora et al., 2009; Wolff & Grundmann, 1992). Unaware of the TVI, as it has not yet been described or illustrated in any textbook of anatomy, some surgeons may have unexpectedly encountered the muscle during surgical procedures. Surgeons who have previously experienced difficulties in surgical procedures within the femoral area may need to consider whether the presence of the TVI was contributory.

As mentioned above, reliable information regarding the anatomical and morphological features has been described by several researchers and confirms the existence of this previously undiscovered muscle ingeniously hidden in the intermuscular region. However, a full understanding of the TVI is incomplete without the additional information needed in order to understand the ontogeny, function, and clinical significance of the muscle. An investigation of the embryology of this area may give us further understanding of the development of the muscular system and the kinetic mechanism of the extensor apparatus at the knee joint. We were able to first identify the TVI using serial tissue sections of human embryos, containing a sufficient number of specimens included in the Kyoto Collection of Human Embryos and Fetuses. The aim of our report is to elucidate the ontogeny of embryonic TVI and to help determine the implications of this unobtrusive muscle.

2. MATERIALS AND METHODS

2.1. Samples

Samples for the present study were collected from normal embryos (with no abnormal external appearance) stored in the Kyoto Collection of Human Embryos and Fetuses owned by Kyoto University, harvested by abortions for social and economic reasons in accordance with the Japanese Eugenic Protection Law until 1995 and the Maternal Protection Law of Japan since 1996. Each specimen had been sliced to a thickness of 10 μm and stained with hematoxylin and eosin. Further information about the procedure of collections has been described in previous reports (Yamaguchi & Yamada, 2018). Human embryos are classified into 23 stages, termed Carnegie stages (CS), according to the developmental features of their external appearance (O'Rahilly & F. Müller, 1987; Yamada & Takakuwa, 2012). The samples included in this study were selected from embryos of CS 21, 22, and 23 (18.5–28.7 mm crown‐rump length [CRL]) that possessed distinguishable quadricep muscles with no defects in their lower limbs. Thirty serial sections from 15 human embryos were observed histologically using a light microscope (Table 1). This study was approved by the Ethics Committee of the Graduate School of Medicine and Faculty of Medicine, Kyoto University (approval no. R0316).

TABLE 1.

Specimens for this study

	No. of cases	Specimen ID
CS21	5	#940, #2021, #2314, #7316, #19393
CS22	5	#10444, #3098, #10642, #12246, #15768
CS23	5	#4381, #9005, #9026, #10273, #12481

2.2. 3D reconstruction

First, to reconstruct a 3D model, including five muscular components of the thigh extensor and the femur, serial sections of a CS 23 embryo (#9026) were digitized with a flatbed scanner (VS120 Virtual Slide Microscope, OLYMPUS). The scanned images were saved in VSI format files. Then, the images were converted into TIFF format using ImageJ software (Schneider et al., 2012). Finally, each muscle of each slide was segmented and stacked to render a 3D model with Amira 6.5.0 software (FEI, Hillsboro).

3. RESULTS

In CS 21, the TVI was not found in any of the specimens (Table 2). The TVI was first observed in CS 22 embryos, in seven out of 10 limbs, and was observed as a thin crescent mass of myocytes between the VL and VI. In CS 23, the TVI was found bilaterally in all specimens, with the exception of one. The one limb without the TVI had an indistinct mass of myocytes where the TVI should exist, as observed in other CS 23 embryos, but this mass had not separated from the VL or VI.

TABLE 2.

No. of TVI, classification into vi‐ or vl‐type in each CS

	No. of TVI	No. of vl‐type	No. of vi‐type
CS21	0	0	0
CS22	7	6	1
CS23	9	6	3
Total	16	12	4

There was no difference in the incidence of the TVI between the right and left limbs. We could not identify any aponeurosis and tendinous structures in any specimens; only muscle bellies could be identified. Additionally, no distinguishable insertions nor origins of the TVI were observed.

On careful observation, the muscle bellies diverged from either the VL or VI muscle in all cases. We classified them accordingly into two groups, “vl‐type” and “vi‐type”, with respect to whether the TVI muscle belly was separated from the VL or VI. Figure 1 shows each sample of both types histologically (a, b) and by using the schema (c, d). According to our classification, vl‐type was present in 12/16 limbs (75%) and vi‐type in 4/16 limbs (25%) (Table 2). Regarding the vasculature of the thigh, the femoral artery was identified; however, the distal branches and the feeding arteries were not observed to be connected to the TVI.

FIGURE 1.

Histological sections and three‐dimensional (3D) reconstruction of the TVI. The TVI observed in CS23 embryos (#9026 and #10273). (a, b) Histological section (hematoxylin & eosin staining) of the vl‐type (#9026) and vi‐type (#10273), respectively. (c, d) Schema of the quadriceps and the TVI based on (a, b) respectively. (e–g) 3D reconstruction from serial sections (#9026). (e) Anterior view of the right thigh. (f) Posterior view of the right thigh. (g) Anterior view of the right thigh without the VL. VM: Vastus medialis (red in 3D) VI: Vastus intermedius (blue in 3D) VL: Vastus lateralis (yellow in 3D). TVI: Tensor vastus intermedius (green in 3D) RF: Rectus femoris (not shown in 3D)

In the 3D reconstructed model (Figure 1, e–g), the TVI was smaller and flatter than the other muscles. It was observed between the VL and VI, and was located in the middle of the thigh, which was more distal than previously reported in adults.

In summary, embryonic TVI begins to develop in CS 22, which is later than the other quadriceps muscles. All specimens were sorted into two groups according to variations of muscular fusion, that is, whether the TVI muscle belly diverged from the VL or VI.

4. DISCUSSION

4.1. Timing of appearance of the TVI

After the first report on the TVI by Grob et al., several researchers have reported the anatomical and morphological features of the muscle. However, no literature exists on the development of the TVI during the embryonic or fetal period, likely due to the difficulty in obtaining appropriate samples in these developmental phases. The Kyoto Collection, owned by the Congenital Anomaly Research Center of Kyoto University, is the largest collection of human embryos worldwide and now stores over 500 sets of serial sections of normal embryos. In addition, a recent work on the imaging of embryos and fetuses stored in the Kyoto Collection has begun using several different imaging devices, including magnetic resonance imaging (MRI), episcopic fluorescence image capture (EFIC), and X‐ray computed tomography (CT) (Yamada et al., 2018; Yamaguchi et al., 2018). However, serial tissue sections are still superior in terms of resolution and precision when inspecting the local microanatomy of each organ. Because of this, we started with histological observations first in order to identify rudimentary TVI. The present study found that the TVI appears in CS 22, indicating that the muscle is already present during the human embryonic stage. According to one report about the development of human limb muscles (Diogo et al., 2019), the four conventional components of the quadriceps femoris are divided from one mass of myocytes, and are completely separated until CRL 18‐mm embryos (corresponding to CS 20–21 embryos). In our findings, while all embryos in CS 21 already had four divided components (as Diogo et al. previously described), none had developed the TVI at this time. Given these findings, we consider that the TVI begins to divide from the VL or VI after the quadriceps femoral has completed its separation. Previous phylogenic studies on the homology among vertebrates have indicated that the VL, VI, and vastus medialis of humans are derived from an unseparated, single reptilian muscle (Diogo & Molnar, 2014). The process of separation of each quadriceps muscle during human embryo development, shown in the present study, is likely to represent a reproduction of the evolutionary history of these muscles, where they have differentiated from a common origin. Furthermore, our observation implies that the TVI possibly has the same origin as other muscles. However, our study was not able to elucidate why the TVI originates from two different embryonic muscles, the VL and VI. Recent evolutionary developmental studies hypothesize that anatomical divergence from homologous structures is caused by a complex interaction between functional, phylogenic, and ontogenic factors (Diogo & Molnar, 2014). Thus we suppose that further studies on the development of the TVI using older fetuses, focusing on its nerve or vessel structure and its kinetic function, may help us to solve this question. Due to the difficulty in making a proper serial section from a specimen larger than approximately 3 cm, the means in which to study these features in larger samples are limited. We are currently developing additional studies using imaging techniques adaptable for both embryos and fetuses and hope that these may give a more comprehensive understanding of the developmental process of this muscle.

4.2. TVI classification in embryos and adults

While the TVI muscle belly was clearly identified in CS 22 and 23 in embryos, no aponeurosis and tendinous structures were observed. We classified our specimens into two groups, vl‐type or vi‐type, depending on whether the TVI belly appeared to separate from the VL or VI. Our result showed that 75% of all specimens was vl‐type and 25% was vi‐type. Notably, our present classification in embryos, is quite different from the classification in adults used by Glob et al., because different features of the TVI were focused on in each study. While Grob et al. emphasized the aponeurotic conjunction of the TVI to adjacent muscles in order to classify the subtypes, our study focused on the histological findings of TVI bellies because of the lack of aponeurotic structure in the embryonic period. We suspect that both vl‐type and vi‐type eventually become Independent‐type as they continue to develop after this period, or become VL‐type or VI‐type if they only partially divide; an incomplete division from both of the VL and VI could induce Common‐type. We hypothesize that VL‐type or VI‐type classification used by Grob et al. may be already determined during the embryonic period, even though not all tissues are fully developed until this period. If the ratio of vl‐type and vi‐type in embryos coincided with that of VL‐type and VI‐type in adults, it would support our hypothesis, however, there is not enough evidence currently to confirm this. In order to elucidate the origin of the morphological variations of the TVI, observation of continuous sample groups from embryos and fetuses to adults is required, along with a larger sample size.

4.3. Development of tendons

To classify the TVI into anatomical subtypes in adults, the extent of aponeurotic or tendinous fusion is important; however, the mechanism of aponeurosis and tendon development is poorly understood. Recent studies in mice embryo suggest that muscles and tendons develop independently in early embryonic period; in later phase, they begin to interact each other, suggesting that tendinous maturation requires the presence of muscle (Tozer & Duprez, 2005).

One hypothesis regarding the development of the TVI tendon suggests that it grows up synergistically in response to the muscular activity of the VI if the TVI regulates the motion of the VI as Grob et al. have referred. Unfortunately, it is not yet known in detail when and how the VI develops as fetal movement increases, because no methods have been established to individually quantify the activity of each extensor muscle. One limitation of our study is that the specimens used were limited to a very narrow range of gestational age. To demonstrate how aponeurosis and tendinous structure of the TVI develop, further studies of a broader range of gestational age including more developed fetuses are needed.

4.4. Clinical significance of the TVI

Proper motion of the knee is maintained by the mechanical and dynamic balance of the quadriceps tendons, multiple supporting ligaments, and numerous other anatomical factors (Amis, 2007; Andrikoula et al., 2006). Therefore, minimizing the damage to ligaments or tendons around the knee during surgery could lead to a decrease in the incidence of some surgical complications. Waligora et al. (2009) stated that a precise investigation of structural variations in tendon insertions into the patella may help avoid patellar joint instability after total knee arthroplasty. We believe that, in addition to the discovery of the TVI, more detailed studies of how it develops would also be helpful for further understanding of the complicated structure of the tendons around the knee.

The intended focus of our study was not only on the muscle itself, but also on the vascular supply, as there are some surgeries in which surgeons would benefit from awareness of variations in the vasculature around the TVI. Grob, Ackland, et al. (2016) have described that the TVI of adults is mainly supplied by the superior or transverse branch of the lateral circumflex femoral artery (LCFA), although no direct vascular supply into the TVI was detected in our study on embryos. There are two flaps, the VL flap and the anterolateral thigh (ALT) flap, elevated in the location anatomically close to the TVI and supplied by branches of the LCFA. Both are myocutaneous or fasciomuscular flaps that utilized for the reconstruction of soft tissue defects after head and neck malignancy or limb injury. The VL flap receives the blood supply from the ascending branch of LCFA, originating from the deep femoral artery, and is then raised up from between the VL and VI. Wolff and Grundmann (1992) pointed out in their report that there were several cases with arterial variation whose proximal branches seemed to be concealed by “muscle fibers between the VL and VI”; this description may imply the existence of the TVI. The ALT flap is usually supplied from the descending branch of the LCFA, which runs adjacent to the aponeurosis of the TVI within the proximal one‐third of the thigh in MRI studies on adult cadavers (Grob, Ackland, et al., 2016; Grob et al., 2017). It is well known that there are several vascular variations in branches from the LFCA (Lakhiani et al., 2012), and a lack of knowledge on vascular variations, which could also include an absence of a certain branch, can interfere with proper surgical procedures and even cause a flap elevation to fail. We suppose that vascular variations in this region may be complicated by the diversity of multi‐layered aponeurosis which may include the TVI. Therefore, we expect that a comprehensive survey of the development of the TVI and its relevant arteries might provide a new insight into the origin of the complicated vasculature in this area and potentially help surgeons avoid flap handling errors.

5. CONCLUSIONS

Our findings revealed that the TVI first appears in CS 22 in early human embryonic development. Embryonic TVI can be classified into vl‐type and vi‐type, based on whether myocytes cluster seem to separate from the VL or VI. Clinically, an accurate knowledge of the TVI may help orthopedists and plastic surgeons to prevent surgical complications related to these anatomical considerations and variations. Further investigations of these features in later fetal phase could help more precisely elucidate the manner in which the TVI muscle develops during the gestational age and could lead to novel insights on the anatomical and functional significance of this new muscle.

CONFLICT OF INTEREST

The authors declare no conflicts of interest associated with this manuscript.

AUTHOR CONTRIBUTIONS

RK and SY designed the study. RK performed the observations and acquired the data. NU performed part of the observation and wrote the manuscript. NU, RK, YY, and SY contributed to data interpretation. IT and SY took part in critical revision of the manuscript. All authors read and approved the final manuscript.