Received: 27 September 1999 Abstract Object: Histopathologicalobservations and biochemical analy-sis of sutural bones in nine patientswith craniosynostosis were com-pared with control subjects of thesame age range. Methods: Micro-scopic examination in craniosynosto-sis showed formation of an activeosseous front, with higher osteoblas-tic activity than in controls. Bio-chemical analysis revealed highercalcium, phosphorus, alkaline phos-phatase, phospholipids and chon-droitin sulphate-A contents in sutural bones of the same patients.Conclusions: The present study systematically establishes a pre-mature increase in osteogenesis inthe sutures of craniosynostosis pa-tients.

Keywords Craniosysnostosis · Cranial suture · Osteoblasts

Child’s Nerv Syst (2000) 16:564–568© Springer-Verlag 2000 O R I G I N A L PA P E R

A. PathakB.K. SandhuB.D. RadotraN. KhandelwalR. NathV.K. Kak

Histopathological and biochemical changes in the sutural region in craniosynostosis

Introduction

Etiologically and pathogenetically, craniosynostosis is aheterogeneous disorder [6] leading to premature fusionof one or more cranial sutures. Skull growth after closureof a single suture was described in 1851 by Virchow[11]. Despite recent reports of a genetic aetiology of thedisorder [22, 34, 35], overall disturbances in craniofacialgrowth in craniosynostosis patients need to be fully un-derstood, as the problem seems to be quite complicated[12, 16].

Since the underlying cause is elusive, surgery remainsthe mainstay of treatment [10, 14, 20, 24, 27, 29, 32]. In-terestingly, this condition has also been noted in primates[9]. Though the skull base has been the focus of primary

pathology, there are suggestions that it can also be foundelsewhere [15, 31]. Experimental studies have been car-ried out in an attempt to understand the biomechanics ofcraniofacial growth [1, 3, 5, 13, 17, 28]. It has been sug-gested that cranial sutures in craniosynostosis have thecapacity to compensate by depositing bone asymmetri-cally along the edges, but no systematic studies havebeen performed on this aspect in human subjects.

The present study was aimed at investigating the his-topathological and biochemical changes occurring in thesutural regions in craniosynostosis patients. The objec-tives of the study were:

a) To study the histopathological changes in suturalbones in craniosynostosis with special reference to os-teoblastic activity, and

A. Pathak (✉ ) · V.K. KakDepartment of Neurosurgery, Postgraduate Institute of Medical, Education and Research, Chandigarh 160 012, Indiae-mail: medinst@pgi.chd.nic.inFax: +91-172-540401

B.K. Sandhu · R. NathDepartment of Biochemistry, Postgraduate Institute of Medical Education and Research,Chandigarh 160 012, India

B.D. RadotraDepartment of Histopathology, Postgraduate Institute of Medical Education and Research,Chandigarh 160 012, India

N. KhandelwalDepartment of Radiodiagnosis, Postgraduate Institute of Medical Education and Research,Chandigarh 160 012, India

b) To study the calcium, phosphorus, alkaline phospha-tase, phospholipids and chondroitin sulphate-A contentof sutural bones in craniosynostosis.

Materials and methods

Nine craniosynostosis patients between 3 1/2 and 18 months ofage (mean 8.8 months) and eight control subjects between 1 dayand 3 years of age (mean 9.8 months) were included in the study.The extent of sutural involvement and the cranial morphologywere evaluated by 3-D CT scans of the head. Estimation of serumcalcium, phosphorus, alkaline phosphatase and creatinine was per-formed in all patients and control subjects to exclude any systemicabnormality. Bones excised from the involved sutures during theoperative procedures performed for craniosynostosis were studiedby histopathological examination and biochemical analysis. Con-trol subjects were patients undergoing neurosurgical operativeprocedures for other lesions, and the tissue made available in thecourse of craniotomy over the corresponding sutural region wassimilarly studied. Tissue sections were stained with haematoxylin-eosin for routine histopathological examination and Masson tri-chrome stain to highlight the collagen. The sutural bones wereanalysed for their calcium content (estimated by the method ofTrinder), phosphorus content (estimated by the method of Fiskeand Subarrow), alkaline phosphatase (estimated by the method ofKing and King), phospholipids content (estimated by the methodof Bartlett) and chondroitin sulphate-A (estimated by the methodof Scott).

Results of biochemical analysis were divided into two groupsby patients’ ages, viz. (i) 0–12 months and (ii) 1–10 years, andwere then compared according to the involved sutural regions(sagittal, coronal and anterior fontanelle).

Results

Histopathological changes

The growing edge of the bone in sutural regions in con-trol group patients was well defined with a clear demar-cation between calcified tissue and surrounding fibrousstroma. Osteoblasts formed a distinct border at the inter-phase between the growing bone and collagen tissue(Fig. 1). An active osseous front with increased osteo-blastic activity was observed in the sutural regions in pa-tients of craniosynostosis (Fig. 2). At higher magnifica-tion, calcification was seen to proceed in a diffuse man-ner with migration of osteoblasts, unlike the well-de-fined lining seen in controls. The advancing osseousfront had islands of osteoid tissue laid in a labyrinthinepattern, giving an impression of impending osseoustransformation (Fig. 3).

Biochemical changes

Biochemical analysis revealed definitely higher contentsof calcium, phosphorus, alkaline phosphatase, phospho-lipids and chondroitin sulphate-A in sutural bones fromthe craniosynostosis patients than in such bone from the

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Fig. 1 Photomicrographs showing a sharp border at the interphasebetween collagen and growing bone in a control subject. (H & E,×140)

Fig. 2 An irregular osseous front showing increased osteoblasticactivity in a craniosynostosis subject. (H&E, ×140)

Fig. 3 Irregular islands of new formed osteoid at the interphasebetween collagen and growing bone (H&E, ×55)

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control group patients (Figs. 4–8). Serum calcium, phos-phorus, alkaline phosphatase and creatinine levels, how-ever, were found to be normal in all subjects.

Discussion

Craniosynostosis has a wide spectrum of clinical pre-sentations, but the underlying pathophysiology remainsin the realms of mystery. Early diagnosis and promptcorrective surgical procedure(s) remain the current man-agement of choice. However, syndromic craniosynost-oses pose greater management difficulties than the non-syndromic variety, owing to other associated problemsin syndromic types, which demand priority in manage-ment.

The skull vault enlarges by way of appositionalgrowth at the sutural joints. Little is known about the de-velopmental biology of this process. Recent reports inthe literature point to various genetic derangements asso-

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Fig. 4 Calcium content (mg/g of ash) of sutural bone from cranio-synostosis patients and controls

Fig. 5 Phosphorus content (mg/g of ash) of sutural bone from cra-niosynostosis patients and controls

Fig. 7 Alkaline phosphatase content (KAU/100 mg of alkali pro-tein) of sutural bone from craniosynostosis patients and controls

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Fig. 8 Chondroitin sulphate-A content (µg/100 g of organic bone)of sutural bone from craniosynostosis patients and controls

Fig. 6 Phospholipid content (mg/g of dry bone) of sutural bonefrom craniosynostosis patients and controls

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ciated with this disorder [2, 19, 21–23, 30, 33, 34]. Mu-tations of the MSx2, FGFR1, FGFR2, FGFR3 andTWIST genes yield new insights into the biogenesis ofcranial sutures [36]. However, this condition is usuallynot associated with any generalised disorder of bone me-tabolism, as reflected in the serum biochemical and hor-monal profiles.

Whatever the causative factor, the end result is mani-fested in the form of localised sutural osteogenesis, asalso observed in the present study. This was histological-ly corroborated by demonstration of an active osseousfront produced by migrating columns of osteoblasts inthe formative bony matrix. Biochemical findings in in-volved sutural regions endorse the above finding bydemonstrating higher levels of the constituents of osteo-genesis, i.ei.e. calcium, phosphorus, phospholipids, alka-line phosphatase and chondroitin sulphate-A, than con-trols.

Osteoblasts cultured from an involved sutural regionhave shown altered indices of cellular mechanism bothin the basal state and in response to provocative agents[25]. The involved osteoblasts produced significantlymore osteocalcin and less alkaline phosphatase. This isin contrast to the in vivo situation in the present study,where biochemical analysis showed an increased alka-line phosphatase content in the involved sutures.

Molecular mechanisms have been thought to inducetissue interaction of the dural cells with sutural cells by

means of growth-factor-mediated pathways [4, 8]. Therole of osteoblasts and osteoclasts have also been studied[26, 36].

Attempts at understanding the sutural biology andcorrelating events in craniosynostosis with long-termhistological observations of sutural life cycle calls intoquestion the significance of various layers in a sutureand their etiological and pathogenetical heterogenicity[7]. In an affected suture, craniosynostosis starts at a sin-gle point and spreads along the suture, to various extentsin different subjects. This suggests that it is not always apansutural event.

The present preliminary study attempts to providequalitative and quantitative measures of the overall in-crease in the process of osteogenesis which takes placein sutural regions in craniosynostosis. The underlyingcausative factors that accelerate the process of biochemi-cal and histological changes prematurely have yet to beidentified. Recent studies demonstrate elevated levels ofgrowth factor(s) in the sutures of patients with cranio-synostosis [18]. However, it remains to be ascertainedwhen and how this enhancement of osteogenesis occurs.The invasive nature of the osteoblasts demonstrated inthe present study also demands further study on the func-tion and maturation of these cells. The development of agenetic animal model of craniosynostosis would allowan insight into the pathogenetic mechanism of this disor-der using molecular techniques.

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