In the present study, the question of whether a relatively non-specific epithelial requirement exists for membrane bone formation within the maxillary mesenchyme was investigated. Organ rudiments from embryonic chicks of three to five days of incubation (HH .18 –25) were enzymatically separated into the epithelial and mesenchymal components. Maxillary mesenchyme (from embryos HH 18 –19) which in the absence of epithelium will not form bone was recombined with epithelium from maxillae of similarly aged embryos (homotypic-homochronic recombination) and of older embryos (HH 25) (homotypic-heterochronic recombination). Heterotypic recombinations were made between maxillary mesenchyme (HH 18 –19) and the epithelium from wing and hind-limb buds (HH 19 –22). Recombinants were grown as grafts on the chorioallantoic membranes of host chick embryos. Grafts of intact maxillae, isolated maxillary mesenchyme, and isolated epithelia from the maxilla, wing-, and hind-limb buds were grown as controls. The histodifferentiation of grafted intact maxillae was similar to that in vivo; both cartilage and membrane bone differentiated within the mesenchyme. Grafts of maxillary mesenchyme (from embryos HH 18 –19) grown in the absence of epithelium formed cartilage but did not form membrane bone. Grafts of maxillary mesenchyme (from embryos HH 18 –19) recombined with epithelium in homotypic-homochronic, homotypic-heterochronic, and heterotypic tissue combinations formed membrane bone in addition to cartilage. These results indicate that maxillary mesenchyme requires the presence of epithelium to promote osteogenesis and that this epithelial requirement is relatively non-specific in terms of type and age of epithelium.

Previous studies have shown that epithelial-mesenchymal interactions are influential in promoting membrane bone formation within mesenchymal tissue. It has been shown for the developing maxilla (Tyler, 1978), mandible, (Tyler & Hall, 1977), and skull (Schowing, 1968 a, b, c;,Benoit & Schowing, 1970) of the chick that the mesenchyme requires the presence of an epithelium during a specific embryonic period early in development in order for subsequent membrane bone formation to occur. Removal of the epithelium during this time period prevents osteogenesis within the isolated mesenchyme. In the systems studied, the presence of the epithelium ceases to be required by the mesenchyme several days prior to the actual onset of osteogenesis. In the developing chick maxilla, epithelial influences are required through Hamburger & Hamilton (1951) stage (HH) 22 ( days of incubation). By HH 23 (4 days of incubation), removal of the maxillary epithelium will not prevent osteogenesis within the mesenchyme. The actual onset of ossification within the maxilla does not occur until HH 35 ( days of incubation) (Tyler, 1978).

The present study examines whether or not the epithelial influence required by maxillary mesenchyme through HH 22 is specific for a particular type or age of epithelium. A similar study on the chick mandible indicates that the epithelial requirement for bone formation within the mandibular mesenchyme is relatively non-specific with respect to the type of epithelium that will promote mandibular osteogenesis, but that the age of the epithelium is an important factor in determining whether the epithelium will be influential in promoting osteogenesis.

In the present study, tissue recombinations were implemented to test the influence of heterochronic and heterotypic epithelia on the genesis of membrane bone within the maxillary mesenchyme. Maxillary mesenchyme was isolated from its epithelium at a time when an epithelial influence is required for osteogenesis, and the mesenchyme was recombined with maxillary epithelium from similarly aged embryos (homotypic-homochronic recombination) and with maxillary epithelium from embryos beyond the stage at which an epithelial influence is required for maxillary osteogenesis (homotypic-heterochronic recombination). In heterotypic recombinations, isolated maxillary mesenchyme was recombined with epithelia from the wing and hind-limb buds. These epithelial regions normally do not participate in promoting membrane bone formation. Recombinations were grown as grafts on the chorioallantoic membrane of host chick embryos.

The results indicate that the epithelial requirement for osteogenesis within the chick maxilla is relatively non-specific and differ to a certain extent from those reported for the chick mandible (Hall, 1978).

Tissue preparation

Eggs from the common chicken (Gallus domesticus, White Leghorn) were incubated in a Leahy, forced-draft incubator at 37·5 ± 1°C and 57 ± 2% humidity. Chick embryos from eggs incubated for three to five days were staged according to the Hamburger and Hamilton (1951) staging (HH) series, and organ dissection was carried out in Tyrode’s solution. Maxillae from embryos HH 18 –19 and HH 25 ( and 5 days of incubation), and wing and hind-limb buds from embryos HH 19 –22 ( days incubation) were used in the study.

Separation of the epithelium from the mesenchyme of an organ was achieved enzymatically. Organs were placed in a 3% trypsin-pancreatin solution (3:1 (w/w) in calcium- and magnesium-free Tyrode’s solution) at 4°C for 45 min. Following enzymatic treatment, the loosened epithelium was removed from the mesenchyme by manipulation with a small-bore pipette and finely sharpened tungsten needles. Separated tissues were placed in a solution of Tyrode’s and fetal calf serum (1:1, v/v) which served to inactivate any residual enzymatic solution in the tissues, and tissues were stored in the Tyrode’s-serum solution until use.

Grafting procedures

Intact maxillae, intact wing and hind-limb buds, the isolated epithelial and mesenchymal components of maxillae, isolated epithelia from the wing and hind-limb buds, and maxillary mesenchyme recombined with epithelia from the maxilla or from the wing or hind-limb buds were grafted to the chorioallantoic membrane of host chick embryos that had been incubated for eight or nine days. As a control to determine whether the enzymatic treatment interfered with tissue differentiation, grafts were made of intact maxillary processes that had been enzymatically treated without subsequent mechanical tissue separation.

Intact organs and isolated tissues were placed on Millipore filter discs (black; 5 mm diameter; 0·45 μm porosity; 125–150 μm thick; obtained from Millipore Filter Corp., Bedford, Massachusetts). The filters served as supports for the explanted tissues and facilitated localization of the graft at the time of harvesting. In tissue recombination experiments, ultra-thin Millipore filter discs (white; 5 mm diameter; 0·45 μm porosity; 25 ± 5 μm thick) were used to allow observation of the tissues by transmitted light during tissue manipulations. To recombine epithelial and mesenchymal tissues, the mesenchyme was placed on the filter and allowed to adhere to the filter; the epithelium was then positioned as a flattened sheet over the mesenchyme. In certain instances, the mesenchyme was positioned on top of the epithelium; this, however, was a less successful method of achieving direct contact between the tissues over a large surface area. In most instances, recombined tissues were placed in a CO2-humidified incubator for one to two hours prior to grafting; this allowed adhesion of the component tissues prior to any further manipulations.

Tissues on their Millipore filter discs were placed on the chorioallantoic membrane of host chick embryos such that the grafted tissues were in direct contact with the host tissue. The host embryos were then further incubated for eight days.

Histological procedures

Grafts were fixed in Bouin’s fluid, dehydrated in a graded series of alcohol, cleared in toluene, and embedded in paraffin blocks. The paraffin blocks were sectioned at 5 μm on a Sorval JB-4 rotary microtome. Mounted sections were stained either with van Gieson’s stain and alcian blue (pH 2·5–3·0) (Wilsman and VanSickle, 1971) or with hematoxylin, eosin, and alcian blue (pH 2·5 – 3·0) (Pearse, 1960).

The results are based on a total of 131 grafts: of these, 14 were of intact rudiments, 15 were of isolated maxillary mesenchyme, 10 were of enzymatically treated intact maxillae, 14 were of isolated epithelia, 17 were of homotypic-homochronic recombinations, 13 were of homotypic-heterochronic recombinations, 22 were of heterotypic recombinations with wing-bud epithelium, and 26 were of heterotypic recombinations with limb-bud epithelium.

Intact maxillary processes

The histodifferentiation of intact maxillary processes excised from embryos HH 18 –19 and HH 25 and grown as chorioallantoic membrane grafts was similar to that reported for the maxilla in vivo (Tyler, 1978). Bony trabeculae representing the elongate membrane bones of the maxilla differentiated within the maxillary mesenchyme, and cartilage, a precursor to the quadrate, an endochondral bone, differentiated usually in close association with membrane bone (Fig. 1, Table 1). The epithelium differentiated into a stratified squamous epithelium consisting of a basal layer of mitotically active cuboidal-to-columnar cells, one to two intermediate cuboidal cell layers, and one to three outer squamous cell layers (Fig. 2). The greater number of cell layers occurred in grafts of maxillae excised from embryos HH 25. In the aboral region of the maxilla, feather germs were distinguishable (Fig. 3). The feather germs were in the hump stage of development in grafts of maxillae excised from young embryos (HH 18-19) and were in the elongation phase of development in grafts of maxillae excised from older embryos (HH 25).

Table 1

Skeletogenesis in intact maxillae and isolated maxillary mesenchyme grafted to the chorioallantoic membrane

Skeletogenesis in intact maxillae and isolated maxillary mesenchyme grafted to the chorioallantoic membrane
Skeletogenesis in intact maxillae and isolated maxillary mesenchyme grafted to the chorioallantoic membrane
Figure 1–6

Figs. 1 –2. Photomicrographs of a section through a graft of an intact maxillary process from a HH-19 embryo grown on the chorioallantoic membrane for 8 days. Cartilage (c), derived from the quadrate, has differentiated near membrane bone (b), and a portion of the membrane bone can be seen in Fig. 2 (arrow) to be in close association with the oral region of the maxillary epithelium (ep). The graft, supported by a Millipore filter (mf), is surrounded by host tissue (ht) from the chorioallantoic membrane. Hematoxylin, eosin, and alcian blue, × 51 and × 95, respectively. Fig. 3. Photomicrograph of a section through the aboral region of the grafted maxilla shown in Fig. 1. Feather germs (fg), shown in longitudinal section, are in the hump stage of development, × 184.

Fig. 4. Photomicrograph of a section through a graft of maxillary mesenchyme isolated from its epithelium at HH 19 and grown on the chorioallantoic membrane for 8 days. Cartilage (c) has differentiated within the mesenchyme, but membrane bone did not form, (ht designates host tissue from the chorioallantoic membrane which surrounds the graft.) Alcian blue and van Gieson’s stain, × 95.

Fig. 5. Photomicrograph of a section through a graft of maxillary mesenchyme isolated from its epithelium at HH 25 and grown on the chorioallantoic membrane for 8 days. Membrane bone (b) has formed in addition to cartilage (c). Host tissue (ht) from the chorioallantoic membrane surrounds the graft. Hematoxylin, eosin, and alcian blue, × 95.

Fig. 6. Photomicrograph of a section through a graft of a maxillary process, removed from its embryo at HH 19, that was enzymatically treated without subsequent tissue separation and then grown on the chorioallantoic membrane for 8 days. Histodifferentiation is similar to that of the grafted intact maxillary process shown in Fig. 1. (b, c, and ep designate membrane bone, cartilage, and epithelium, respectively.) Alcian blue and van Gieson’s stain, × 95.

Figure 1–6

Figs. 1 –2. Photomicrographs of a section through a graft of an intact maxillary process from a HH-19 embryo grown on the chorioallantoic membrane for 8 days. Cartilage (c), derived from the quadrate, has differentiated near membrane bone (b), and a portion of the membrane bone can be seen in Fig. 2 (arrow) to be in close association with the oral region of the maxillary epithelium (ep). The graft, supported by a Millipore filter (mf), is surrounded by host tissue (ht) from the chorioallantoic membrane. Hematoxylin, eosin, and alcian blue, × 51 and × 95, respectively. Fig. 3. Photomicrograph of a section through the aboral region of the grafted maxilla shown in Fig. 1. Feather germs (fg), shown in longitudinal section, are in the hump stage of development, × 184.

Fig. 4. Photomicrograph of a section through a graft of maxillary mesenchyme isolated from its epithelium at HH 19 and grown on the chorioallantoic membrane for 8 days. Cartilage (c) has differentiated within the mesenchyme, but membrane bone did not form, (ht designates host tissue from the chorioallantoic membrane which surrounds the graft.) Alcian blue and van Gieson’s stain, × 95.

Fig. 5. Photomicrograph of a section through a graft of maxillary mesenchyme isolated from its epithelium at HH 25 and grown on the chorioallantoic membrane for 8 days. Membrane bone (b) has formed in addition to cartilage (c). Host tissue (ht) from the chorioallantoic membrane surrounds the graft. Hematoxylin, eosin, and alcian blue, × 95.

Fig. 6. Photomicrograph of a section through a graft of a maxillary process, removed from its embryo at HH 19, that was enzymatically treated without subsequent tissue separation and then grown on the chorioallantoic membrane for 8 days. Histodifferentiation is similar to that of the grafted intact maxillary process shown in Fig. 1. (b, c, and ep designate membrane bone, cartilage, and epithelium, respectively.) Alcian blue and van Gieson’s stain, × 95.

Isolated maxillary mesenchyme

In explants of maxillary mesenchyme isolated from its epithelium during early development (HH 18 –19) and grown as a graft in the absence of its epithelium, cartilage differentiated, but membrane bone did not form (Fig. 4, Table 1). These results confirm those of an earlier study (Tyler, 1978) indicating that the presence of an epithelium is required at this stage for maxillary membrane bone formation. In grafts of maxillary mesenchyme isolated from its epithelium at a later stage of development (HH 25), membrane bone formed in addition to cartilage (Fig. 5, Table 1). This confirms an earlier report (Tyler, 1978) that at this stage in development, an epithelial influence is no longer necessary for promoting maxillary osteogenesis.

The histodifferentiation of intact maxillae (HH 18 –19) that were enzymatically treated without subsequent mechanical tissue separation and then grown as chorioallantoic membrane grafts was similar to that of grafted maxillae that had not been enzymatically treated (Fig. 6), indicating that the enzymatic separation techniques do not cause irreparable damage to the component maxillary tissues.

Homotypic recombinations of maxillary mesenchyme and epithelium

In homotypic tissue recombinations, maxillary mesenchyme from young embryos (HH 18 –19) was recombined with maxillary epithelium from similarly aged embryos (homotypic-homochronic recombination) and with maxillary epithelium from older embryos (HH 25) (homotypic-heterochronic recombination). The position of the tissues with respect to one another was not according to their original orientation.

In grafts of homotypic-homochronic recombinants, the histodifferentiation of the recombined tissues was similar to that of grafted intact maxillae. Membrane bone formed within the mesenchyme in addition to cartilage (Table 2), and the degree of epithelial differentiation was similar to that of grafted intact maxillae of a similar cumulative age (initial age + incubation time as graft). Membrane bone formed usually in close proximity to the epithelium. No specificity was exhibited in terms of epithelial region with which bone was associated; bone was found in association with both oral and aboral regions of the maxillary epithelium.

Table 2

Skeletogenesis in maxillary mesenchyme (HH 18 –19) recombined with homotypic and heterotypic epithelium and grafted to the chorioallantoic membrane

Skeletogenesis in maxillary mesenchyme (HH 18 –19) recombined with homotypic and heterotypic epithelium and grafted to the chorioallantoic membrane
Skeletogenesis in maxillary mesenchyme (HH 18 –19) recombined with homotypic and heterotypic epithelium and grafted to the chorioallantoic membrane

In grafts of homotypic-heterochronic recombinants, mesenchymal differentiation was similar to that of grafted homotypic-homochronic recombinants (Table 2). Membrane bone formed usually in close proximity to the epithelium of either the oral or aboral maxillary regions (Fig. 7). Cartilage formed often in close proximity to membrane bone (Fig. 8). Epithelial differentiation in these recombinants was similar to that of grafted intact maxillae with a cumulative age equal to that of the epithelium rather than to that of the mesenchyme of the heterochronic recombinant (Fig. 9).

Figure 7–12

Fig. 7. Photomicrograph of a section through a homotypic-heterochronic recombinant graft. Maxillary mesenchyme, isolated at HH 19, was recombined with maxillary epithelium, isolated at HH 25, and grown on the chorioallantoic membrane for 8 days. Membrane bone (b) has formed in close association with the epithelium (ep) and cartilage is not in the vicinity of the membrane bone. Alcian blue and van Gieson’s stain, × 95.

Fig. 8. Photomicrograph of a section through a homotypic-heterochronic recombinant graft similar to that in Fig. 7. In this graft, cartilage (c) is found in close association with membrane bone (b), and the membrane bone has formed in close proximity to the epithelium (ep). Alcian blue and van Gieson’s stain, × 95.

Fig. 9. Photomicrograph of a section through the graft in Fig. 8 showing the aboral region of the maxillary epithelium. Feather germs (fg), shown in longitudinal section at their base and in transverse section more distally, are in the elongation stage of development and are more advanced than those shown in Fig. 3. Alcian blue and van Gieson’s stain, × 184.

Fig. 10. Photomicrograph of a section through a heterotypic recombinant graft. Maxillary mesenchyme, isolated at HH 19, was recombined with wing-bud epithelium, isolated at HH 21, and grown on the chorioallantoic membrane for 8 days. Mesenchymal histodifferentiation is similar to that of homotypic recombinant grafts as shown in Fig. 8. Epithelial differentiation is similar to that of grafted intact wing buds with a similar cumulative age. Feather germs (fg) are in the elongation stage of development, (c and b designate cartilage and membrane bone, respectively.) Alcian blue and van Gieson’s stain, × 95.

Fig. 11. Photomicrograph of a section through a heterotypic recombinant graft similar to that shown in Fig. 10 except that the chondrogenic region of the mesenchyme was not included in the explant. Membrane bone (b) has formed in close proximity to the epithelium (ep) in the absence of cartilage, (ht and mf designate host tissue and Millipore filter, respectively.) Hematoxylin, eosin, and alcian blue, × 184.

Fig. 12. Photomicrograph of a section through a graft of maxillary epithelium (ep), isolated at HH 19 and grown in the absence of its mesenchyme on the chorioallantoic membrane for 8 days. The epithelium, underlaid by fibroblasts of host tissue (ht) origin, has differentiated into a stratified squamous epithelium. Regions of the epithelium have formed epithelial whorls (arrow) rather than remaining as a flattened sheet. Feather germs are not present within the graft. Hematoxylin eosin, and alcian blue, ×372.

Figure 7–12

Fig. 7. Photomicrograph of a section through a homotypic-heterochronic recombinant graft. Maxillary mesenchyme, isolated at HH 19, was recombined with maxillary epithelium, isolated at HH 25, and grown on the chorioallantoic membrane for 8 days. Membrane bone (b) has formed in close association with the epithelium (ep) and cartilage is not in the vicinity of the membrane bone. Alcian blue and van Gieson’s stain, × 95.

Fig. 8. Photomicrograph of a section through a homotypic-heterochronic recombinant graft similar to that in Fig. 7. In this graft, cartilage (c) is found in close association with membrane bone (b), and the membrane bone has formed in close proximity to the epithelium (ep). Alcian blue and van Gieson’s stain, × 95.

Fig. 9. Photomicrograph of a section through the graft in Fig. 8 showing the aboral region of the maxillary epithelium. Feather germs (fg), shown in longitudinal section at their base and in transverse section more distally, are in the elongation stage of development and are more advanced than those shown in Fig. 3. Alcian blue and van Gieson’s stain, × 184.

Fig. 10. Photomicrograph of a section through a heterotypic recombinant graft. Maxillary mesenchyme, isolated at HH 19, was recombined with wing-bud epithelium, isolated at HH 21, and grown on the chorioallantoic membrane for 8 days. Mesenchymal histodifferentiation is similar to that of homotypic recombinant grafts as shown in Fig. 8. Epithelial differentiation is similar to that of grafted intact wing buds with a similar cumulative age. Feather germs (fg) are in the elongation stage of development, (c and b designate cartilage and membrane bone, respectively.) Alcian blue and van Gieson’s stain, × 95.

Fig. 11. Photomicrograph of a section through a heterotypic recombinant graft similar to that shown in Fig. 10 except that the chondrogenic region of the mesenchyme was not included in the explant. Membrane bone (b) has formed in close proximity to the epithelium (ep) in the absence of cartilage, (ht and mf designate host tissue and Millipore filter, respectively.) Hematoxylin, eosin, and alcian blue, × 184.

Fig. 12. Photomicrograph of a section through a graft of maxillary epithelium (ep), isolated at HH 19 and grown in the absence of its mesenchyme on the chorioallantoic membrane for 8 days. The epithelium, underlaid by fibroblasts of host tissue (ht) origin, has differentiated into a stratified squamous epithelium. Regions of the epithelium have formed epithelial whorls (arrow) rather than remaining as a flattened sheet. Feather germs are not present within the graft. Hematoxylin eosin, and alcian blue, ×372.

Heterotypic recombinations of maxillary mesenchyme and epithelium from the wing- and hind-limb buds

Heterotypic tissue recombinations were made between maxillary mesenchyme isolated from young embryos (HH 18 –19) and epithelium isolated from wing and hind-limb buds of embryos HH 19 –22, and recombinants were grown as chorioallantoic membrane grafts. Mesenchymal differentiation in each type of recombinant was similar to that of homotypic recombinants irrespective of the source (wing or hind-limb bud) or the intial age (HH 19-22) of the epithelium (Table 2). Cartilage formed within the mesenchyme of the explant and membrane bone was generated usually in close proximity to the epithelium (Fig. 10). In two instances, the chondrogenic region of the maxillary mesenchyme was not included in the explant, and in these grafts membrane bone formed in close association with the epithelium in the absence of cartilage (Fig. 11).

Epithelial differentiation in heterotypic recombinants was similar to that of grafted intact wing and hind-limb buds. The epithelium became a stratified squamous epithelium consisting of a cuboidal germinative cell-layer, one to two intermediate cuboidal cell layers, and one to two outer squamous cell layers. Feather germs in the elongation phase of development were distinguishable and were at the same level of development as those of grafted intact wing and hind-limb buds of a similar cumulative age (Fig. 10). Differences between wing and hind-limb-bud epithelium were not detected.

In approximately 27% of all recombinant grafts, close association between epithelium and mesenchyme was not maintained; in these instances, membrane bone failed to form within the mesenchyme though cartilage did form. Epithelial-mesenchymal contact and consequent osteogenesis were maximized experimentally by blanketing the already substrate-adherent mesenchyme with the epithelium and placing recombined tissues in a CO2-humidified incubator for 1 to 2 h prior to grafting.

Isolated epithelium from the maxilla, wing-bud, and hind-limb bud

Isolated epithelia from the maxilla (HH 18 –19 and 25) and from the wing- and hind-limb buds (HH 19 –22), grown as chorioallantoic membrane grafts in the absence of their mesenchyme, became underlaid by host fibroblasts from the chorioallantoic membrane and achieved a limited degree of differentiation (Fig. 12). In grafts of each different type of epithelium, the epithelium differentiated into a stratified epithelium consisting of a germinative cuboidal cell-layer and one to five outer cell layers which graded from cuboidal to squamous. Feather germs were not observed, nor were skeletal elements (either cartilage or bone) found within the host tissue associated with the grafted epithelium.

It has been shown in a previous study (Tyler, 1978) and confirmed in this study that during early development the presence of an epithelium is a requirement for ensuing genesis of membrane bone within the mesenchyme of the embryonic chick maxilla. The results further indicate that this epithelial requirement is relatively non-specific. In homotypic recombinations, it was shown that re-establishing the original orientation of the maxillary epithelium with respect to its mesenchyme was not necessary for osteogenesis; membrane bone formed within the mesenchyme of the recombinants irrespective of the epithelial orientation. The results from heterotypic recombinations established that maxillary mesenchyme does not specifically require maxillary epithelium to promote osteogenesis; other types of epithelia which in normal development are not associated with membrane-bone-forming mesenchyme (epithelium from the wing- and hind-limb buds) were shown to be capable of promoting osteogenesis within maxillary mesenchyme. From heterochronic recombinations, it was shown that the response of maxillary mesenchyme to epithelium is not restricted to a specific age of epithelium; epithelium removed from maxillae after the time during which the epithelium is required for osteogenesis (isolated at HH 25) is still capable of promoting osteogenesis in maxillary mesenchyme isolated from young embryos (HH 18 –19).

These results differ to a certain extent from those of a similar study on osteogenesis in the embryonic chick mandible (Hall, 1978); in both studies, however, it is concluded that the epithelial requirements for mesenchymal membrane bone formation are relatively non-specific. In the study on mandibular osteogenesis, hind-limb-bud epithelium was found to promote membrane bone formation in mandibular mesenchyme isolated at an early stage of development (HH 18) (Hall, 1978) as was shown for maxillary mesenchyme in the present study. In contrast to our results, however, the results in the mandibular study indicate that neither wing-bud epithelium nor homotypic(mandibular) epithelium promotes osteogenesis within mandibular mesenchyme (isolated at HH 18) in either homochronic or heterochronic recombinations. The epithelial requirements for osteogenesis in the developing chick mandible, therefore, appear to be more restrictive than those in the developing chick maxilla. Whether the results reflect intrinsic differences in the two osteogenic systems or whether the differences between the two studies are a reflection of the different techniques used for growing the tissues (organ culture, Hall, 1978; chorioallantoic membrane graft, this study) is still to be determined. It has been shown that both organ culture and the chorioallantoic membrane of host embryos promote normal histogenesis of intact organ rudiments; however, the two environments have been shown to differ in the amount of tissue growth that each supports and in the type of organ morphogenesis that occurs within each (Tyler and Hall, 1977). Further studies of maxillary and mandibular osteogenesis, therefore, are being made to determine the osteogenic potential of maxillary tissue recombinations in organ culture and mandibular tissue recombinations grown as chorioallantoic membrane grafts.

In earlier histological studies of membrane bone formation it was suggested, based on the proximity of cartilage to the mandibular membrane bones, that cartilage is necessary for membrane bone formation (Frommer & Margolies, 1971). This suggestion has yet to be substantiated, and results from the present study, in which membrane bone formed within maxillary mesenchyme in the absence of cartilage in two heterotypic recombinant grafts, indicate that the presence of chondrogenic centers (beyond HH 18) is not required for maxillary osteogenesis. This conclusion is supported by results from an earlier study (Tyler, 1978).

The results from grafts of epithelium separated from the maxilla, wing,- and hind-limb buds and grown in the absence of its mesenchyme confirm earlier reports (Tonegawa, 1973; Tyler & Hall, 1977; Tyler, 1978) that host fibroblasts from the chorioallantoic membrane are sufficient to maintain a germinative cell layer within an epithelium and to support a limited degree of epithelial histodifferentiation. That the host mesenchymal tissue did not participate in. feather formation suggests that there are specificity requirements for the type of mesenchyme that will support feather formation within an epithelium. This suggestion is supported by other recombination studies of feather- and non-feather-forming tissues (e.g. Rawles, 1963; Dhouailly, 1978). The fact that the grafted epithelium, though capable of promoting osteogenesis in maxillary mesenchyme, did not induce membrane bone formation in the host tissue associated with it indicates that the presence of epithelium, though a requirement for maxillary membrane bone formation, is not a sufficient condition for inducing bone formation in normally non-osteogenic mesenchyme.

In summary, the results of this study indicate that in the developing chick maxilla, reciprocal epithelial-mesenchymal interactions are necessary for normal histodifferentiation and that the epithelial requirement for genesis of membrane bone within maxillary mesenchyme is relatively non-specific with respect to the source and age of the epithelium.

The authors are grateful to Mr David C. Warner for his skilled technical assistance. This investigation was supported by Research Grant 1 R23 DEO4859-02 from the National Institute of Dental Research.

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