Paper - The early development of the knee joint in staged human embryos

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Gardner E. and O'Rahilly R. The early development of the knee joint in staged human embryos. (1968) J. Anat., 102(2): 289-99. PMID 5643844

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Ronan O'Rahilly J. Anat. (1968), 102, 2, pp. 289-299

Here is the link to the paper PDF version.

See also: Haines RW. The development of joints. (1947) J. Anat. 81, 33-55.

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The Early Development of the Knee Joint in Staged Human Embryos

Ronan O'Rahilly
Ronan O'Rahilly

With 16 figures

Ernest Gardner and Ronan O’rahilly

Departments of Anatomy, Wayne State University, Detroit, Michigan, and Saint Louis University, Saint Louis, Missouri, and Department of Embryology, Carnegie Institution of Washington, Baltimore, Maryland


During the last two decades a wealth of information about the development of bones and joints has been published. Such studies are now especially pertinent because of the increasing interest in congenital anomalies of the skeleton. In any discussion of development during the embryonic period, some standard of age or degree of development must be available by which different embryos can be compared. A single criterion, such as C.R. length, has long been used as an index of age, but any one criterion is subject to individual variation. These difficulties can be overcome by staging the embryos, a procedure which has been employed for a number of non-human vertebrates; for example, various amphibians and notably the chick (Hamburger & Hamilton, 1951). Each stage is characterized by a number of external, morphological criteria, and it is possible to discuss and compare differentiation and growth interms that are not confused by variations that inevitably result when a single criterion is used.

Owing chiefly to the work of Streeter (1951), it is now possible to discuss human embryonic development in terms of stages (the so-called ‘developmental horizons’). Streeter divided the embryonic period of development (the first 7-8 weeks after fertilization) into 23 stages. Each stage was identified by a number of external and internal characteristics, and each embryo at a given stage has a similar degree of organization and differentiation to that of other embryos at that stage. In recent years, several studies of the development of the limbs and certain joints in man have been published, using staged human embryos (O’Rahilly, Gardner & Gray, 1956; O’Rahilly, Gray & Gardner, 1957; Gray, Gardner & O’Rahilly, 1957; Gardner, Gray & O’Rahilly, 1959). However, in earlier studies, such as that of Gray & Gardner (1950) on the knee joint, staged material was not available, and a more recent study of the knee joint (Andersen, 1961) also did not make use of staged specimens. It seemed appropriate, therefore, to review the embryonic development of this important joint in staged human embryos.


Thirty-four human embryos of the collection at the Carnegie Institution were used. These serially sectioned embryos had been staged according to Streeter’s ‘developmental horizons’, and ranged from about 5 or 6 weeks of age (time since ovulation) to 7 or 8 weeks (perhaps 9-10 ‘menstrual weeks’). The stains employed were haematoxylin and eosin, alum cochineal, orange G, and ‘azan’. A list of the embryos examined is provided in Table 1.


From previous studies it is known that the lower limb buds first appear during stage 13, that is, in embryos about 5 mm long and about 4 weeks after fertilization. From stage 13 to stage 18, the ectodermal thickening and ridge develop. By about 5 weeks after fertilization, the skeletal parts of the lower limb are beginning to chondrify in proximodistal sequence. By stage 18, chondrification is beginning in the femur, tibia, and fibula, which were already indicated as mesenchymal condensations. The present study begins with stage 18.

Table 1

Table 1. Embryos used in the present study (1968 - Knee)
Streeter’s stage Usual C.R. length (mm) Estimated age in post-ovulatory days (Olivier & Pineau, 1962) No. of embryos examined C.R. lengths (mm)
18 12-17 44 9 11-7, 12-9, 14, 14-4, 14-5, 15, 15, 16-5, 17
19 16-19 47.5 6 16-3, 17, 18, 18-5, 19-1, 21
20 18-23 50.5 9 18, 18-5, 19-5, 20, 20-8, 21, 21, 22, 23
21 22-24 52 5 22, 22-5, 22-5, 22-7, 24
22 23-28 54 3 23-4, 25-3, 25-8
23 27-31 56.5 2 27, 31

Stage 18 (9 embryos; 11-7-17 mm) (approximately 6 postovulatory weeks). The femur, tibia, and fibula had begun to undergo chondrification. The region of the knee joint was represented by a mass of blastemal cells (fig. 1). A differentiating ligamentum patellae could be identified in two of the embryos. In each of these two embryos also, chondrification was somewhat more advanced. During stage 18, the fibula was relatively close to the femur. 1

Stage 19 (6 embryos; 16-3-21 mm) (approximately 7 postovulatory weeks). The femoral condyles were forming, but in the majority of specimens they were mostly blastemal. The blastema intervening between the femur and the tibia was becoming recognizable as a homogeneous interzone.

A definite cellular condensation for the fibular collateral ligament was observed in three specimens and in one of these embryos the tendon of the popliteus could be identified. The ligamentum patellae was clearly present in all specimens and in at least two a cellular condensation for the patella was found (fig. 2). In. one specimen a suggestion of early patellar retinacula was noted. During stage 19, the fibula was relatively close to the femur.

fig. 1. The knee joint is represented by a mass of blastemal cells between the chondrifying femur (Fe) and tibia (Ti). Stage 18. No. 7707. Section 45-2-3. H.-E. Phloxine. x 55.

fig. 2. Lower end of femur. Note the cellular condensation for the patella (arrow). Stage 19. No. 5609. Section 11-1-1. Alum cochineal. x 55.

fig. 3. Femoral condyles and tibia (Ti), together with intervening homogeneous interzone. Stage 20. No. 8226. Section 10-2-4. Mallory ‘azan’. x 55.

fig. 4. Medial condyle of femur and tibial collateral ligament (arrow). Stage 20. No. 462. Section 35-2-1. Alum cochineal. x 100.

In one specimen, a slight suggestion of a cellular condensation for the cruciate ligaments was recorded, but the plane of section was not favourable enough for certain identification.

Stage 20 (9 embryos; 18-23 mm) (approximately 7 postovulatory weeks). The femoral and tibial condyles were clearly evident (fig. 3), although in some specimens they were mostly blastemal and merged with the intervening zone. In the other specimens, however, the condyles were well along in chondrification, thereby defining even more the homogeneous interzone. Also, the chondrifying lateral tibial condyle was beginning to be evident between the femur and the fibula (fig. 5).

Except in one embryo in which it was very faint, the patella was clearly evident as a cellular condensation with characteristic shape and in one instance was almost chondrifying. The fibular collateral ligament (fig. 5) and the tendon of the popliteus (fig. 6) were clearly present, and the tibial collateral ligament (fig. 4) could be identified in some specimens. In at least two specimens, the cruciate ligaments were indicated, and, in at least one, the lateral meniscus. The patellar retinacula were usually identifiable.

Stage 21 (5 embryos; 22-24 mm) (approximately 7% postovulatory weeks). The femoral (fig. 7) and tibial condyles were well defined and mostly cartilaginous. The blastemal interzones showed evidence of a three-layered arrangement. The patella was chondrifying in at least four of the specimens (fig. 8) and patellar retinacula were present in all embryos (fig. 9). The fibular collateral ligament was usually present, together with the tendon of the popliteus, but the tibial collateral ligament could not always be identified. In at least three specimens, an indication of the cruciate ligaments and the laterial meniscus was found (fig. 10). Blood vessels were observed in the peripheral part of the joint.

Stage 22 (3 embryos; 23-4-25-8 mm) (approximately 7%—8 postovulatory weeks). In general, the tibia, fibula, and femur had clear-cut, cartilaginous forms. The chondrogenous layers of the interzones were beginning to meet. The patella was chondrifying, and bone collars were present around the femur and the tibia.

The knee joint resembled that of the adult. Blood vessels were present in its interior. Tendons were clearly differentiated and cellular. The cruciate ligaments were present as cellular, oriented proliferations, and both menisci were identifiable, the cells of the lateral being more definitely oriented. The collateral ligaments were present and were very cellular.

Stage 23 (2 embryos; 27 and 31 mm) (approximately 8 postovulatory weeks). The femur and the tibia had bone collars. The knee joint clearly resembled the adult in form and arrangement (figs. 11, 12). The menisci were clearly defined, and very cellular, with oriented cells (fig. 13). They were somewhat darker than tendons, but nevertheless closely resembled tendons and ligaments. They contained a few collagenous fibres. Nothing in their appearance suggested the presence of fibrocartilage, however. Inwardly, the menisci merged into the femorotibial interzones, which were very thin. Both cruciate ligaments (figs. 12, 14) and both collateral ligaments were clearly identifiable as cellular, oriented structures.

fig. 5. Femur (Fe), tibia (Ti), fibula (fi), and fibular collateral ligament (arrow). Stage 20. No. 462. Section 36-3-4. Alum cochineal. x 100.

fig. 6. Femur, tibia, and tendon of popliteus (arrow). Stage 20. No. 462. Section 34-1-2. Alum cochineal. x 125.

fig. 7. Lower half of femur showing condylar development and, in centre of shaft, marked cartilaginous hypertrophy. Stage 21. No. 8553. Section 96-1-2. H.-E. x 55.

fig. 8. Chondrification in patella (arrow). Stage 21. No. 9614. Section 79-1-4. Mallory ‘azan’. x 100.

Blood vessels were present along the circumferential aspects of the menisci, deep to the ligamentum patellae, around the cruciate ligaments, and in the loose tissue of the posterior part of the joint.

A fibrous capsule distinct from ligaments and retinacula was most uncertain. In the lower part of the joint posteriorly there was a faint proliferation for the capsule, together with a cellular condensation for the oblique popliteal ligament.

In one embryo no joint cavities were present. In the other, in one knee, the loose tissue between the patella and the femur clearly suggested incipient cavitation. In the other knee, a small but definite femorpatellar cavity was present in several consecutive sections (figs. 11, 15). In one knee also, a definite slit-like cavity was present between the medial menisci and the medial femoral condyle. In the other knee of the same specimen a cavity was present anteriorly between the lateral meniscus and the femur (figs. 13, 16). A suggestion of a cavity between the lateral meniscus and the lateral femoral condyles was noted but it could not be said to be definitely present.


In general, the findings of Gray & Gardner (1950) were confirmed, although the present study establishes the sequence and timing of events with much greater precision. The differentiation from a generalized cellular blastema to a joint resembling the adult in form and arrangement occurs in only a relatively few days. No recapitulation exists in the sense that structures characteristic of adult lower forms normally appear as an intermediate phase. Articular structures such as ligaments develop in situ and undergo no migration having phylogenetic significance.

The critical period in the development of human limbs is from about 3 to 6 weeks after fertilization (perhaps 5-8 ‘menstrual weeks’), that is, from just before the limb buds appear until differentiation is well under way. Clinical evidence supports this concept. In the phocomelias which appeared in almost epidemic numbers in Germany in 1959-62, thalidomide being implicated as the teratogenic agent, the critical period appeared to be from 4—6 weeks after conception (6-8 ‘menstrual weeks’). The critical period in the development of the knee joint would roughly correspond to that of the lower limb, but very likely is somewhat shorter in duration.

fig. 9. Medial condyle of femur, quadriceps femoris, and medial patellar retinaculum (arrow). Stage 21. No. 8553. Section 96-2-2. H.-E. x 55.

fig. 10. Femur, tibia, and lateral meniscus (arrow). Stage 21. No. 8553. Section 97-1-3. H.-E. x 100.

fig. 11. General view of knee joint at end of embryonic period proper. Note femoropatellar cavity, which is shown also in fig. 15, in another section from the same specimen. Stage 23. No. 9226. Section 191-3-5. Mallory ‘azan’. x 55.

fig. 12. General view of knee joint at end of embryonic period proper. Note cruciate ligaments. Stage 23. No. 9226. Section 193-1-5. Mallory ‘azan’. x 55.

fig. 13. Femur, tibia, and lateral meniscus. Stage 23. No. 9226. Section 188-2-2. Mallory ‘azan’ x 100.

fig. 14. Higher-power view of previous section to show early cavity between femur and lateral meniscus. x 200.

fig. 15. Cruciate ligaments. Femur (Fe) above and tibia (Ti) below. Stage 23. No. 9226. Section 185-2-3. Mallory ‘azan’. x 100.

fig. 16. Early femoropatellar cavity. See fig. 11 for general view. Stage 23. N0. 9226. Section 191-3-5. Mallory ‘azan’. x 100. Early development of knee joint 297

The discoid meniscus, which is of some clinical importance, is not an arrested phase of normal development; it must be viewed as a variation from the normal pattern (Gray & Gardner, 1950; Kaplan, 1955).

The importance of studying staged embryos and of having a sufficient number of specimens to assess individual variation is emphasized by certain aspects of a paper by Andersen & Bro-Rasmussen (1961) on the development of joint cavities in the hand and foot. They attempted a critical analysis of the manner and sequence of cavitation in a series of 25 foetuses of which only 3 were less than 61 mm in C.R. length (12, 30 and 48 mm, respectively).

Table 2

Table 2. Comparative sequential development of human knee joint and that of chick embryo (1968 - Knee)
  (The data relating to Gallus domesticus are from O’Rahilly & Gardiner, 1956.)
Feature Homo stage Gallus stage
Lower limb bud 13 17
Ectodermal thickening and ridge 13-18 18-30
Skeletal chondrification 18 27
Femorofibular proximity 18-19 27
Quadriceps insertion 18-19 27
Tibialis anterior in knee 27
Ambiens in knee 29
Homogeneous interzone 19-20 26
Patellar condensation 19-20 29-30
Intracapsular ligaments, including cruciate and menisci 19-20 30
Embryonic movements c. 20 c. early 30’s
Three-layered interzone 21 ?
Chondrification in patella 21-22 36
Diaphysial bone collars 22-23 28-29
Cavitation 23 34

More directly applicable to the present study is a paper by Andersen (1961) on the development of the knee joint. Of 26 foetuses used, 9 were less than 32 mm C.R. (11, 12, 20, 23, 23, 25, 25, 26 and 30 mm, respectively). The author states: ‘The stage of development was related to the crown—rump length, although the data thus obtained cannot be said to be particularly exact for scientific purposes.’ He nevertheless used the phrase ‘ considerably earlier’ (p. 283) in comparing two unstaged embryos whose crown—rump lengths are different but whose degree of development was similar.

In most respects, Andersen’s findings appear to be similar to those reported by previous investigators (Haines, 1947; Gray & Gardner, 1950; Haines, 1953 ; O’Rahil1y & Gardner, 1956). However, some of his interpretations are decidedly at variance. For example, he reported that from an early stage the menisci consist of fibrocartilage. He appears to have based this belief, at least in part, upon the specificity of the staining reaction with Alcian blue. He stated that ‘ at 23 mm there is a very faint metachromatic reaction in the meniscal prirnordia and a corresponding blue staining with Alcian blue, indicating the formation of a cartilaginous matrix ’. Yet, on the same page, he pointed out that in well-fixed preparations clotted synovial fluid in the joint cavity stains a very bright blue with Alcian blue.

Andersen also raises some pertinent questions with regard to the role of the blastema in the formation of articular structures. However, he seems to attribute more specificity to his histochernical techniques than may be warranted, and until these and other matters are clarified, continued discussion of these questions will yield little of substance.

Although the details of osteogenesis differ considerably between the chick and the human, the general morphogenesis of the elbow and knee joints is closely similar in the two species (O’Rahjlly & Gardner, 1956; Gardner & O’Rahilly, 1962). For purposes of comparison, the chief features observed in the formation of the human and avian knee joints are listed in Table 2, from which it can be seen that the sequence of development is fundamentally similar.


  1. Serial sections of 34 embryos aged 5-8 postovulatory weeks (12-31 mm C.R.) were examined. The embryos had been staged according to Streeter’s "developmental horizons".
  2. The femur, tibia, and fibula had begun to undergo chondrification by stage 18. The condyles and a mesenchymal patella were distinguished at stages 19-20, and the patella had commenced chondrification at stages 21-22. Bone collars began to form in the femur and tibia at stages 22-23.
  3. The following structures became condensed successively: ligamentum patellae (stages 18-19), fibular collateral ligament and tendon of popliteus (19), patellar retinacula and cruciate ligaments (19-20), tibial collateral ligament and menisci (20), and oblique popliteal ligament and articular capsule (23).
  4. A homogeneous interzone became defined between the skeletal elements (stages 19-20), and joint cavitation had commenced in one embryo at the end of the embryonic period proper (stage 23), by which time collagenous fibres were present in the menisci.
  5. In general, the findings of Gray & Gardner (1950) were confirmed, although the sequence and timing of events were established with much greater precision.

This work was aided by grants AM 6705-02 and NB 04426-02 from the United States National Institutes of Health, Public Health Service.


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