Zeitschrift fiir Zellforschung 72, 193--211 (1966)

T H E H I S T I O C Y T E I N H U M A N F O E T A L T I S S U E S

ITS MORPHOLOGY, CYTOCHEMISTRY, ORIGIN, FUNCTION, AND FATE*

HELGE ANDERSEN a n d M. E. MATTHIESSEN

Laboratory of Cyto- and Histoehemistry, Department of Human Anatomy, University of Copenhagen, Denmark

Received December 3, 1965

Summary. Based upon a material comprising human foetuses cytochemicM studies of a widespread type of cell were carried out. Apart from amoeboid mobility the cell is characterized by pinocytotic and phagocytotic activity.

In early development stages these cells are seen intravascularly and penetrating the vessels. Later they are seen in connection with the formation of the vascular epiphyseal cartilaginous canals and in the vitreous body, the synovial joints and dental anlage. Furthermore these cells are seen at the removal of the epithelial cells of the dental lamina and the junctional epithelium in the two palatine processes. The cells concerned are seen also in the deep periosteal layer at the centre of the diaphysis synchronously with the vascularization of the periosteum and prior to the periosteal invasion. Based upon morphology and cytochemistry the theory is advanced that these cells form the chondroclasts and the multinucleated osteoclasts. By contrast, the diaphysial osteoblasts are derived from invading pre-osteoblasts from the cambium-layer of the periosteum.

These cells are also seen along the basal surface of the neural apparatus and invading the brain vesicles.

On the basis of morphology and cytochemistry the cell type is designated a histiocyte and its origin is traced back to primitive leucocytes.

I n p r e v i o u s s tud ies conce rn ing t h e d e v e l o p m e n t of t h e s y n o v i a l jo in t s (AND]~R-

SV, N, 1962 a n d 1964) a n d dec iduous t e e t h (MATTHIESSEN, 1965) an a b u n d a n c e of a

h i s t iocy te - l ike cell t y p e were seen. Th is cell has r e c e i v e d b u t l i t t l e a t t e n t i o n in t h e

p a s t a n d t h e pu rpose of t he p r e s e n t s t u d y was on t h e basis of m o r e r e c e n t cy to-

chemica l m e t h o d s to m a k e an i n v e s t i g a t i o n on t h e m o r p h o l o g y , c y t o c h e m i s t r y ,

o r ig in a n d f u n c t i o n of these cells.

Material and Methods The material comprised 41 human foetuses, removed by Caesarean section in connection

with legal abortion. Based on crown-rump length the material varied from 5,5 mm to 230 mm, corresponding to a menstrual age ranging from the 4th to the 26th week. All measurements were made on unfixed foetuses.

_Fixation. Almost all the foetuses were fixed within 30 min post mortem with the exception of tissues intended for lactic dehydrogenase and NADH 2 cytochrome C reductase investigations, in which cases fresh frozen sections were used. Ice-cold fixatives were used, as also the fixation per se was carried out at 0 4 ~ C.

The following fixatives were applied:

1. Buffered, neutral 4 per cent formaldehyde was used for the fixation of tissues for various amino acid reactions and for fixation of the hydrolytic enzymes: alkaline phosphatases, acid phosphatases, and unspecific AS-esterase as well as for fixation of leucyl-aminopeptidase. The

* This work has been supported by grants from the Danish State Research Foundation, the Rask-Orsted Foundation and The Association for the Aid of the Crippled Children, New York.

194 HELGE A~I)~RSE~ and M. E. MATTItIESSEN:

time of fixation was 30 min for alkaline phosphatases, whereas unspecific AS-esterase was fixed for 3 h and acid phosphatases for 3 to 18 h. Tissues intended for aminopeptidase reaction were fixed for 15 to 30 min. With a view to the denmnstration of enzymes, the tissue was transferred after fixation to two shifts of ice-cold arabic gum-sucrose, whereby the enzyme activity is preserved and the subsequent cutting on the freezing microtome is facilitated.

2. Ethanol-formalimacetic acid (LILLI~, 1954). This fixative was used for morphologic investigations, acid mueopolysaccharides, glycogen, and nucleic acids.

Sectioning. For the detection of the hydrolytic enzymes and aminopeptidase, frozen sections of 6 microns were used, whereas fresh frozen sections of 6 to 8 microns were used for the demonstration of dehydrogenase. As regards the latter, the sections were placed soon after sectioning in a deep-freezer until incubation took place. For the remaining reactions paraffin- embedded sections of 4 to 6 microns were applied.

Cytochemica l reaction,s

I. React ions for acid mucopolysacchar ides

1. Metachromatic staining with 0.1 per cent toluidine blue in 30 per cent ethanol (KRAM~A~ and WI~DRVM, 1955).

2. Staining with 0.1 per cent Alcian blue in 3 per cent acetic acid (MowRY, 1956). Subse- quently some sections were counterstained with ordinary PAS or with a van Gieson staining. Prior to the treatment with the acid media, Alcian blue was converted into the insoluble Monastral Fast Blue by treatment for 2 to 3 h with 0.3 per cent sodium carbonate (McMANvs and MOWRY, 1960). When handling embryonic tissue, the latter step is important (AN])ERSEN, 1964).

I I . Glycogen and other PAS-posi t ive substances

1. Periodic acid-Schiff (PEARSE, 1960). Although stated by KUGLER and WILKINSO~ (1961) that celloidine coating prior to periodic acid has no influence upon the preservation of glycogen, it is the present authors' experience that such coating is important when dealing with embryo- nic material (ANDERSEN, 1964). As recommended by KASTnN (1960), a Schiff reagent advoca- ted by B~GER and DF~ LAMATER was employed.

Control: Malt~se and saliva, digestion before celloidine coating.

I I I . Nucleic acids

i. Gallocyanin-chromalum staining (EI~ARSO~, 1951 ; PAXK~BERG, 1962). Control : Digestion with ribonuclease (Fluka AG).

IV. Special amino acids

1. Bennett's 1-(4-ehloromercuriphenylazo)-2-naphthol (Mercury Orange) for sulphydryl groups (BENNETT and WATTS, 1958) with ethanol as a solvent.

Control: Mercaptide blockade with phenyl mercuric chloride. 2. Millon reaction for tyrosine (modified by ANDERSEN, 1964). Control : Iodination of the tyrosine side chain (P~RS]~, 1960). 3. Yasuma and Itchikawa's ninhydrin-Schiff method for protein-bound NH~-groups

(PEASE, 1960; KAST~, 1962). Control : Avoiding deamination with ninhydrin before Schiff's reagent.

V. Enzymes

1. The naphthol-AS-BI-phosphate method for alkaline phosphatase (BuRsTONE, 1958). [ncubation periods: 1---10 min at room temperature.

2. The naphthol-AS-BI-phosphate method for acid phosphatase (BURSTON~, 1958). In- cubation periods: 4--45 min at 370 C. (When incubating for osteoclasts the procedure was also carried out at temperatures from 0 ~ ~ C).

3. The naphthol-AS-acetate method for esterase (GoMoRI, 1952; BURSTONE, 1958). Incu- bation periods: 10--45 rain at room temperature.

Histiocyte in Human Foetal Tissues 195

4. The L-leucyl-naphthyl-amidehydrochloride method for aminopeptidase (BuRsTONE, 1962). Time of incubation: 2--15 min.

Control/or all/our enzymes: Sections incubated avoiding substrates. 5. The Thomas and Pearse method (1961) for lactic dehydrogenase(s) (LDH), using Nitro-

BT as a hydrogen acceptor. To avoid the "Nothing Dehydrogenase" reaction the method was modified according to ANDnRSE~ (1965).

6. The NADH 2 cytochrome c reductase method (THOMAS and PEARSE, 1961; BURSTONE, 1962).

Observations

Morphology. After f ixa t ion with Lfllie 's e thanol - formal in-ace t ic acid, the mor- phological deta i ls will be seen much more d i s t inc t ly t han af ter fo rmaldehyde f ixa t ion alone. I n the l a t t e r case the cells in quest ion will appea r heav i ly shrunken wi th pycnot ic nuclei, in pa r t i cu la r in regions where the cells are ex t r eme ly vacuo- la ted. W i t h regard to morpho logy the cell t ype examined differs c lear ly f rom the

Fig. 1. Two histiocytes (h) in the highly metachromatic loose infrapatcllar tissue of the knee joint. Note strong vacuolization in the cytoplasm, v vessels. Staining: Toluidine blue. • 720

sur rounding o rd ina ry s ta r - shaped or sp indle-shaped mesenchymal s t roma cells, being larger wi th a b u n d a n t cy toplasm, which is often heav i ly vacuola ted , especial ly if the cells are found in t issues r ich in acid mucopolysacchar ides (Fig. 1). I n add i t ion to clear vacuoles, there are somet imes large heav i ly basophi le granules, which are f requen t ly found to appea r s ingly in the cell (Figs. 2 - -3 ) , and which can a t t a in d imensions corresponding to half the size of the cell nucleus. However , the mos t f requent f inding is several small basophi le granules sca t t e red in the cy top lasm in be tween the clear vacuoles.

I n form the cell nucleus varies f rom round to oval and conta ins one or two d i s t inc t nucleoli. The nuclear m e m b r a n e will of ten - - in pa r t i cu la r a t l a te r s tages of deve lopmen t - - p resen t an i nden t a t i on a t one side, which is seen more d i s t inc t ly a f te r mercury-o range staining, which produces a s t rong reac t ion in the nuclear membrane . I n the cases where the cell conta ins cy top lasmic clear vacuoles of var ious dimensions, the nucleus will of ten be more condensed and smal ler wi th inden ta t ions corresponding to sur rounding cy top lasmic vacuoles (Fig. 1).

196 HELGE ANDERSEN a n d M. E . MATTnIESS~N:

Fig. 2. One of the histiocytes (h) situated in a blood vessel with nucleated red blood cells, e endothelium cells. Note strong basophil inclusion in the cytoplasm of the histiocyte. Staining: Toluidine blue. • 740

:Fig. 3. Dense, basophil cytoplasmic inclusion in a histiocyte (h) laying outside a vessel. Metachromatic staining with toluidine blue. • 740

Histiocyte in Human Foetal Tissues 197

In the loose mesenchyme the shape of the cells varies greatly. They will often present pseudopodia as a sign of amoeboid mobility. Also the appearance of the pseudopodia varies greatly from several small rounded cytoplasmic processes; to a single long club-shaped process.

Cytochemistry. After chromalum-gallocyanin staining the cell presents a mo- derate cytoplasmic basophilia in evidence of a minor content of RNA. In the cases where the cell is par t ly filled with cytoplasmic clear vacuoles, this basophilia appears as fine reticular networks in between the vacuoles. These networks stain metachromatically with toluidine blue. With both staining techniques the reaction disappears after ribonuclease digestion.

The fine cytoplasmic granules present positive PAS-reaction and reaction for free SH-groups with mercury-orange. Furthermore, they present positive reaction for protein-bound NH2-groups with ninhydrin-Schiff, whereas the Millon test for tyrosine shows only diffuse cytoplasmic staining.

Although the large clear cytoplasmic vacuoles are found primarily in regions where the cell is surrounded by heavily acid mucopolysaccharide-containing tissues, these vacuoles never react with tolnidine blue and Alcian blue as a manifestation of these substances being present in the vacuoles.

The cell presents an extremely strong activity for acid phosphatase and un- specific AS-esterase. As regards both these reactions it applies that the final reac- tion product appears to be nodular with a uniform cytoplasmic distribution. A corresponding nodular appearance of the final reaction product is seen in the re- action for leueyl-aminopeptidase, whereas the cell does not show any signs of the presence of alkaline phosphatase. With regard to the dehydrogenase activity, the cell is not very easily distinguished from the surrounding mesenchymal stroma cells.

Glycogen was never observed in the cytoplasm of the cells in question. Reviewing the material at disposal it may be stated that morphologically and

metabolically the cell type studied is clearly different from the surrounding ordi- nary mesenchymal stroma cells, the latter only presenting an extremely modest metabohc activity.

Origin. During the early stages of development the cells in question are seen intravascularly (Figs. 4---5) and in the perivascular connective tissue, but never in non-vascularized mesenchyme. Frequently the cells are observed when they are about to pass the vascular wall by means of their amoeboid mobility (Fig. 6). Near the vessels small groups of the cells may be seen, and on the whole it applies - - also in later stages of development - - tha t the cells discussed here occur most fre- quently perivascularly. The cells are seen not only in the secondary mesoderm, but also in derivatives of the pr imary mesoderm when the latter is vascularized. Hence, a few of the cells are seen in the chorionic villi at a stage when the total length of the embryo is only 5.5 mm (Fig. 7).

Function and fate. In the vascular mesenchyme the cell presents a vivid amoe- boid mobility and shows signs of vivid pinocytosis, indicated by the appearance of cytoplasmic vacuoles of various dimensions, which present a pronounced acti- v i ty for acid phosphatases and for unspecific AS-esterase. The most pronounced pinocytotic activity seems to appear when the cell is found in tissues with a high concentration of acid mucopolysaccharides. Hence, this is seen in the chorionic villi of the placenta, in the synovial tissues and the synovial cavity, in the enamel

198 HELGE ANDERSEN and M. E. MATTnI]:SS~:

Fig. 4. Two histiocytes (h) in a vessel (v) in <lense mesenchyme of an embryo at 12 mm crown-rump length. Tolui dine blue staining, x 600

Fig. 5. Three histiocytes (arrows) inside a vessel (v). Another one outside the vessel. Toluidine blue staining. • 740

reticulum, the dental pulp, and the vitreous body, as well as along the basal surface of the neural apparatus in the loose tissue from which the subarachnoid space is

Histiocyte in Human Foetal Tissues 199

Fig. 6. Two his t iocytes (arrows) pass ing through the wall of a vessel (t,). Two other h i s t iocy tes laying outs ide th vessel. Tohfidine blue s ta ining. / 740

Fig. 7. His t ioeyte (h) in the mesenchymal , highly metachromat ic central cord of a p lacentar viUus belonging an embryo at 5,5 m m length. Toluidine blue s ta ining. • 340

derived. In all these regions the cell type concerned is found in fairly large numbers, which greatly exceed the numbers found in the ordinary vascular mesenchyme.

2 0 0 HELGE ANDERSEN a n d M. E . MATTHIESSEN:

Fig. 8. Numerous histiocy~es (some marked with arrows) showing strong activity of AS-estcrase in the vitreous body (cv) along the ramification of the hyaloid artery, r retina. ~Naphthol-AS-acetate method for esterase. • 220

In the synovial tissue they first occur synchronously with the vascularization of this tissue, whereas they never appear in the interzonal layer where at a later stage the joint cavity is formed (ANDERSEN, 1962). As soon as the joint cavity is formed, the cells are seen in great numbers in the superficial synovial layers, where they are often so closely packed that they may resemble single-layered or multi- layered epithelium. They are also present in the joint cavity proper, where they are surrounded by the synovia which stains intensely metachromatically and with Alcian blue (ANDERSEN, 1964).

In the vitreous body they are found in particular along the branches of the hyaloid artery (Fig. 8), where they form up to 50 per cent of the total number of cells seen in the vitreous body. The cells found in the peripheral parts of the vitre- ous body are mainly of this type.

During the development of the two palatine processes the cell occurs concur- rently with the appearance of vessels in the oral parts of the palatine processes. After fusion of the processes has taken place, the cells are seen to be densely packed on the epithelial wall ot the site of fusion (Fig. 9), where they contribute to the disintegration of this junctional epithelium (ANDI~lCSEI~ and M A T T H I E S S E N ,

1966). When during the development of the chondral skeleton the cpiphyseal carti-

lages have reached a certain dimension, the hitherto avascular hyaline cartilages

Histiocyte in Human Foetal Tissues 201

Fig. 9. 5Tumerous histiocytes (arrows) in close contact with the junctional epi thel ium (ep) of the fused palatine processes. I n some places the epithelium has been broken down. Naphthol-AS-acetate method for esterasc. • 330

become vascularized and are gradually penetrated by vascular mesenchymal canals. These canals are derived from the perichondrium and begin as local groups of the above cells, which come in close contact with the cartilaginous matrix, whereupon chondrolysis takes place. As the canal extends further into the cartilage, large

202 HELGE ANDERSEN and M. E. MATTttIESSEN:

Fig. 10. Collection of his t iocytes (h) in the front pa r t of an epiphyseal , vascu la r canal (vc) in the head of humerus . ec epiphyseal carti lage. Naphthol -AS-BI-phosphate method for acid phosphatase. • 160

number of these cells are constantly seen at the front part of the canal (Fig. 10) and along the lateral walls. At the same time the adjacent areas exhibit signs of lysis of the cartilaginous matrix accompanied by release of chondrocytes (ANDElC- SEX, 1962).

Even before the periosteal invasion into the diaphysis takes place, the cells concerned are seen in close association with the vessels in the deep cellular part of the periosteum (the cambium layer) on a level with the centre of the diaphysis (Fig. 11). After invasion into the diaphyseal cartilage has taken place, the cells are found in the recently formed marrow cavity lying in the opened lacunae of the hypertrophic cartilage cells (Fig. 12) and more scattered along the invading vessels. Furthermore the deep periosteal layers contain cells of the type mentioned, lying in close association with the periosteal bone collar and sometimes with long cyto- plasmic processes extending into the collar (Fig. 13). In addition to the cells discus- sed above, star-shaped and spindel-shaped cells are seen in the periosteal tap. The appearance of these cells is similar to that of the pre-osteoblasts in the deep cellular periosteal layers, and they present a strong alkaline phosphatase activity similar to that of the pre-osteoblasts. This activity becomes almost as intense as that of the osteoblasts which gradually appear along the spicules of calcified cartilaginous matrix which are seen in the centre of the diaphysis and which have not undergone chondroclastic lysis. Gradually as multinucleated osteoclasts appear in the dia- physeal ossification centre, these cells exhibit the same cytochemical character- istics as the above mononucleated cells with regard to acid phosphatases, unspeci- fic AS-esterase, leucyl-aminopeptidase, and lactic dehydrogenase - - NADH~

Histioeyte in Human Foetal Tissues 203

Fig. 11. A hist iocyte (h) in the deep camb ium layer of the per ios teum of the humerus before the periosteal invasion of the diaphyse. ]p f ibrous periosteum, po preosteoblasts , o osteoblasts, b periosteal bone collar, c hyper t rophic

cartilage, v vessel. Staining: Toluidine blue. • 600

eytochrome c reductase, however so that the activity is slightly higher for acid phosphatase and for the system of LDH-NADH 2 cytochrome c reductase. As regards acid phosphatase, incubation at 0 4 ~ C is required, if the exact enzymatic localization is to be determined, since the reaction occurs almost momentaneously at 37 o C. At 0--40 C a very early enzymatic activity is seen in the "striated border". In the multinucleated osteoclasts large cytoplasmic vacuoles are seen which con- tain particles that stain intensely inetachromatically and with Alcian blue. In a few exceptional cases mitotic activity is seen in the multinucleated osteoclasts, whereas cytoplasmic indentations are often seen in these cells, indicating that they may have arisen by the fusion of a number of mononucleated cells.

In connection with the endesmal ossification of the mandible, the type of cell examined occurs in great numbers (Fig. 14) around the primary spongiosa at the time when multinucleated osteoclasts are not yet to be seen. Also here they first appear in a perivascular position.

In connection with the germs of the deciduous teeth, and parallel with the development of the bell stage, perivascular cells of the same appearance as those discussed above appear along the external enamel epithelium. Cells of this type are also seen lying more singly in the intercellular substance in the stratum reticulare and in greater numbers along the vessels in the dental pulp (MATTHIESSEN, 1965). When the dental lamina disintegrates and remains as epithehal pearls, a great

Z. Zellforsch., Bd. 72 14

204 HELGE ANDERSEN a n d M. E . MATTHIESSEN:

Fig. 12. Numerous histiocytes (chondroclasts) (some indicated by arrows) in the lacunae of the hypertrophic chondro- cytes (he) in the diaphyseal ossification center of the tibia. Naphtllol-AS-BI-phosphate method for acid phosphatase.

200

Fig. 13. Histiocyte (h) in the deep cellular, periosteal cambium layer of the tibia. Note the long cytoplasmic pro- trusion into the periosteal bone collar (b). c cartilage. Naphthol-AS-BI-phosphate method for acid phosphatase.

• 920

H i s t i o c y t e in H u m a n F o e t a l T i s sue s 205

~'ig. 14. Numerous histiocytes (most of them indicated by arrows) in the vicinity of the mandibular bone anlage (b). oc multinucleated osteoclasts, Naphthol-AS-BI-phosphatase method for acid phosphatase. • 160

Fig. 15. Numerous histiocytes (most of them m~rked with arrows) in close contact with the disintegrating dental lamina (elo). Naphthol-AS-acetate method for esterase, • 200

14"

206 HELGE ANDERSE~ and M. E. MATTHIESSEN:

many of the above cells are seen in close contact with the epithelial cells of the dental lamina (Fig. 15) and with the epithelial pearls. During the early stages of the disintegration, these cells become gradually more numerous from the surrounding mesenchyme towards the dental lamina.

In the vascular mesenchyme, basal to the neural apparatus where the suba- rachnoid space will be formed later, an abundance of the cells concerned is seen lying perivascularly and accompanying the vessels into the walls of the cerebral vesicles. These cells are also seen in the choroid plexus.

Discussion and Conclusions

The nodular cytoplasmic localization of the final reaction products of acid phosphatase, unspecific AS-esterase, and leucyl-aminopeptidase in the cells studied indicates strongly that they contain lysosomes (DE DuvE, 1959), since the enzymes in question - - and in particular the first two of them - - are so common consti- tuents of lysosomes that they are considered the most important characteristics in the cytochemical detection of lysosomes (BARKA and ANDERSON, 1963). This is further supported by the presence of the small PAS-positive granules in the cyto- plasm of the cells, since it is well-known that the lysosomes are surrounded by a lipoprotein membrane which exhibits a PAS-positive reaction.

The variable large clear cytoplasmic vacuoles which are often seen in the cells, suggest that the cells exert a certain pinocytotic or phagocytotic activity, since these vacuoles give reaction for acid phosphatase and unspecific AS-esterase. The vacuoles must presumably be regarded as fused pinocytotic vesicles, to which is added the content of lysosomes, i.e., the phagolysosomes described by STRAUS (1964). In addition the cells present a distinct histiolytic activity, which is most pronounced when the cells are lying in front of the vascular mesenchymal canals in the epiphyseal cartilages. In these parts the adjacent cartilage matrix presents a pronounced reduction in metachromatic reaction, indicating tha t the cartilage matrix is changing concurrently with the release of chondrocytes from their lacunae (ANDnRSE~, 1962). The histiolytic activity is also seen in the palatine processes and at the dental lamina, where the cells are seen in close contact with epithelial ceils, which present signs of degeneration and removal during the subsequent developmental stages.

The abovementioned conditions in connection with the amoeboid mobility of the cells constitute fairly definite characteristics of histiocytes, and it must there- fore be presumed that these cells represent such types of cells during the earliest stages of embryonic development and that their presence in the vascular system during the early embryonic stages as well as their passage through the walls of the vessels strongly indicate that their origin must be traced back to the early primi- tive blood cells belonging to the leucocyte types. This assumption is further sub- stantiated by the fact that the cells appear in the various tissues only when such tissues are vascularized and that they always appear synchronously with such vascularization.

The reason why these cells have at t racted so little attention, must most likely be sought for in the fact tha t most authors have used mainly formaldehyde fixa- tion. This fixative is in itself an excellent medium of the additive type, but it gives poor results because of pronounced shrinkage of the cells, when the tissue is

Histiocyte in Human Foetal Tissues 207

embedded in paraffin and dehydrated subsequent to the formaldehyde fixation. Hence, the cells and their nuclei show a marked shrinkage and it is rather difficult to distinguish them from the surrounding mesenchymal connective tissue ceils - - in particular if they are fixed 1/2--1 h or more post mortem. By contrast, Lillie's ethanol-formalin-acetic acid produces good morphological details, but the best impression of the histiocytes in question is obtained by cutting on the freezing microtome. The preparations used in the present study for enzymatic reactions were embedded in glycerine-gelatine without previous dehydration and shrinkage to about two thirds of the natural size of the cell. The shrinkage resulting from formaldehyde fixation, which is discussed above, is most pronounced if the cells present the variable large cytoplasmic clear vacuoles.

The function of the ceils concerned is fairly distinct in connection with the formation of the vascular epiphyseal cartilaginous canals and with the disintegra- tion of the cells of the junctional epithelium of the palatine processes and the epithelial ceils in the dental laminae as well as transformation of the histiocytes into chondroclasts and osteoclasts in the diaphyseal ossification centres. By con- trast, their function is more obscure in areas where they appear in tissues with a high content of acid mucopolysaccharides such as synovial tissue, joint cavity, the vitreous body, the enamel reticulum, and the dental pulp. In this connection it is of interest to observe that experiments in vitro have shown that histiocytes are at t racted very much by highly viscous solutions (BARNETT, DAVIES and McCoNAILL, 1962). Consequently, the presence of the histiocytes in these tissues which are very viscous on account of the high content of acid mucopolysaccharides, may be a result of the more incidental attraction which these substances exert on the histiocytcs. In a paper from 1965 on esterase activity in guinea pig embryos, BuNo described the presence in the vitreous body of cells with a high esterase activity, without relating these cells to histiocytes. He designated the cells in question "hyaloid cells" and pointed out tha t they had not previously been described in the literature.

Whereas it is universally agreed tha t chondroblasts, chondrocytes, pre- osteoblasts, and osteoblasts are derivatives of the skeletal blastema, there is violent disagreement as to the origin of osteoclasts and chondroclasts. Many theories have been advocated as to the development of osteoclasts, as appears from a recent review of HA~cox (1956). In the present study it is shown that the histiocytes in question do not appear in the skeletal blastema, until it is vascularized from the surrounding ordinary vascular mesenchyme, and that they appear in the skeletal blastema along the vessels concurrently with the ingrowth of the latter. Hence, they appear in the deep perichondreal layer before the vascular epiphyseal carti- laginous canals are formed and in the deep pcriosteal layers (the cambium layer) along the capillaries before the periosteal ingrowth in the diaphysis takes place. Here they may be seen with long cytoplasmic processes extending into the peri- osteal bone collar and they must be presumed to play a role in the penetration of this collar. When the periosteal ingrowth is completed, these cells are seen lying scattered in the pr imary marrow cavity and in groups in the opened lacunae of the cartilage cells. In the same manner the histiocytes are seen in great numbers along the vessels around the mandibular anlage a long time before multinucleated osteoclasts are seen.

208 HELGE ANDERSEN and M. E. M~TT~ESSEN:

Hence, these histiocytes are of extraskeletal blastemal origin and at the same time they are the only type of cells, which may apparently be responsible for the formation of osteoclasts and chondroclasts. In addition to their presence in the vascular epiphyseal cartilaginous canals, they will be seen in the diaphysis subse- quent to the periosteal ingrowth, situated in the lacunae of the hypertrophic chondrocytes when these are opened up by disintegration of the cartilage. As re- gards the formation of the multinucleated osteoclasts, it is stated that the cyto- chemical reactions applied in the present study reveal a marked similarity be- tween the mononucleated histiocytes and the osteoclasts, both of which may also present large cytoplasmic vacuoles, which as regards the osteoclasts often present a granular material staining metachromatically and with Alcian blue. The fairly pronounced reaction for acid phosphatase, which is observed in the "striated border" of the osteoclasts, may be interpreted as an excretion of the cellular con- tent of lysosomes during the breakdown of the bone, as suggested by DE Duv~ (1963), and is in accordance with similar studies carried out by HAND ELMAN, MORSE and IRvI~o (1965). This reaction appears prior to the actual cytoplasmic reaction and is almost momentaneous, and it is therefore not very likely tha t it is an artefact caused by diffusion or nonspecific reaction. Besides, lysosomes have been demon- strated both in histiocytes (STRAUS, 1959) and in osteoclasts (HANcox and Boo- THROYD, 1963).

The general concept that osteoclasts arise by fusion of mononucleated cells, is in agreement with the present study, since we have often observed osteoclasts with deep cytoplasmic indentations corresponding to the interspace between the cell nuclei present. This might indicate an incipient fusion of several mononucleated cells. By contrast, mitotic activity is extremely infrequent in the osteoclasts, and if it occurs it will be in the osteoclasts only, which have two nuclei. This mitotic activity can hardly explain the appearance of the many multinucleated osteoclasts which are observed rather soon in the diaphyseal ossification centre.

Consequently, according to the present study, there must be two clearly distin- guishable cell precursors in the diaphyseal ossification centre, one for the chondro- clasts and ostcoclasts and one for the osteoblasts. The first two being derived from the immigrating histiocytes of extraskelctal-blastemal origin, whereas the osteoblasts are derived from immigrating pre-osteoblasts from the cambium layer of the periosteum (Fig. 16). This conception is clearly inconsistent with YouNG's observation (1962a, b), tha t both osteoclasts and osteoblasts are derived from a common type of cells, which he designated osteoprogenitor cells. On the other hand the present study is in agreement with YOUNG's observation that the osteoclasts are formed by fusion of mononucleated cells, an observation which was further substantiated by FISCHMA~ ~ and HAY (1962), although these authors stated that the osteoclasts are formed by fusion of monocytes. The present study corresponds completely with JEE and NOLAN'S investigation (1962). By injecting bone charcoal in white rabbits these authors reached the conclusion tha t it was histiocytes which were transformed into osteoclasts by fusion. However, the two authors did not decide upon the origin of the histiocytes.

Fairly large problems, however, remain to be solved as to the early differentia- tion processes relating to pre-osteoblasts and osteoblasts. Studies carried out by one of us (ANDERSEN, 1963) show that the periosteal ossification does not begin

Histiocyte in Human Foetal Tissues 209

until vessels have penetrated the perichondrium on the level of the centre of the diaphysis. Till then the entire skeletal blastema has been avascular and has con- tained only two types of cells, chondroblasts and chondrocytes. In this connection it is of interest to note tha t in vitro experiments with embryonic osteoblasts (BAssETT, 1962) show that these cells require a large supply of oxygen to form bone matrix, whereas a small supply of oxygen leads to the formation of cartilage only.

Diagram showing the two types of osteoprogenitor cells. F P fibrous periosteum, PO preosteoblasts, H histioeyte, V vessel, 0 osteoblasts, OC osteoc]ast, CHC chondroclast

Fig. 16.

On the other hand the oxygen supply can not be the only factor in the differentia- tion of the osteoblasts since BASSETT'S experiments show that apart from oxygen the embryonic osteoblasts require a uniform pressure from all directions to be able to form bone matrix, and AND]~I~S]~N (1963) finds that the pr imary endesmal ossification in the clavicle begins centrally in an avascular accumulation of pre- osteoblasts. In addition FELL (1956) states tha t the cartilaginous hypertrophy at the centre of the diaphysis is presumably the stimulus which transforms the perichondrea] cells into osteoblasts. In any case she finds that this hyper t rophy of the chondrocytes in the centre of the diaphysis always sets in prior to the peri- chondreal cell transformation.

However, it must not be forgotten that no mat ter how interesting in vitro cultivation of cells is, these cells are in an artificial environment and are with- drawn from the homeostasis or steady state of the organism, and it can just be emphasized tha t our present understanding of the early cyto-differentiation is extremely incomplete.

210 HELGE ANDERSEN and M. E. MATT~IIESSEN:

The large concentra t ion of the histiocytes basal to the neural appara tus and the invas ion into the walls of the bra in vesicles of these cells synchronously with the ingrowth of vessels agree in a way with the general assumpt ion tha t microglia is developed t rom perivascular mesenchymal cells which invade the central ner- vous system. However, in the present invest igat ion the subsequent development of the microglia has not been studied.

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Dr. HELGE ANDERSEN Cyto- og Histokemisk Laboratorium, Medicinsk-Anatomisk Insti tut Universitetsparken 1, Kopenhagen O, Denmark