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Avascular Necrosis of Femoral Head: A Metabolomic, Biophysical, Biochemical, Electron Microscopic and Histopathological Characterization

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• Published: 06 September 2017

Avascular Necrosis of Femoral Head: A Metabolomic, Biophysical, Biochemical, Electron Microscopic and Histopathological Characterization

• Aswath Narayanan, 
• Prakash Khanchandani, 
• Roshan M. Borkar, 
• Chandrashekar Reddy Ambati, 
• Arun Roy, 
• Xu Han, 
• Ritesh N Bhoskar, 
• Srinivas Ragampeta, 
• Francis Gannon, 
• Vijaya Mysorekar, 
• Balasubramanyam Karanam, 
• Sai Muthukumar V & 
• Venketesh Sivaramakrishnan 

Scientific Reports volume 7, Article number: 10721 (2017) Cite this article

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Abstract

Avascular necrosis of the femur head (AVNFH) is a debilitating disease caused due to the use of alcohol, steroids, following trauma or unclear (idiopathic) etiology, affecting mostly the middle aged population. Clinically AVNFH is associated with impaired blood supply to the femoral head resulting in bone necrosis and collapse. Although Homocysteine (HC) has been implicated in AVNFH, levels of homocysteine and its associated pathway metabolites have not been characterized. We demonstrate elevated levels of homocysteine and concomitantly reduced levels of vitamins B6 and B12, in plasma of AVNFH patients. AVNFH patients also had elevated blood levels of sodium and creatinine, and reduced levels of random glucose and haemoglobin. Biophysical and ultrastructural analysis of AVNFH bone revealed increased remodelling and reduced bone mineral density portrayed by increased carbonate to phosphate ratio and decreased Phosphate to amide ratio together with disrupted trabeculae, loss of osteocytes, presence of calcified marrow, and elevated expression‎ of osteocalcin in the osteoblasts localized in necrotic regions. Taken together, our studies for the first time characterize the metabolomic, pathophysiological and morphometric changes associated with AVNFH providing insights for development of new markers and therapeutic strategies for this debilitating disorder.

요약

대퇴골두 무혈성 괴사(AVNFH)는 

알코올, 스테로이드 사용, 외상 후 또는 불분명한 (특발성) 병인으로 인해 발생하는 

쇠약성 질환으로 주로 중년층에 영향을 미칩니다. 

임상적으로 AVNFH는 

대퇴골두로의 혈액 공급 장애로 인해 뼈가 괴사하고 무너지는 것과 관련이 있습니다. 

호모시스테인(HC)이 AVNFH와 관련이 있지만, 

호모시스테인 및 관련 경로 대사산물의 수준은 특성화되어 있지 않습니다. 

우리는 

대퇴골두 무혈성괴사 환자의 혈장에서 호모시스테인 수치가 상승하고 

동시에 비타민 B6와 B12 수치가 감소하는 것을 확인했습니다. 

또한

대퇴골두 무혈성괴사 환자는 

혈중 나트륨과 크레아티닌 수치가 상승하고, 

무작위 포도당과 헤모글로빈 수치가 감소했습니다. 

대퇴골두 무혈성괴사 뼈의 생물물리학적 및 초구조적 분석 결과, 

탄산염 대 인산염 비율의 증가, 

인산염 대 아미드 비율의 감소와 함께 

섬유주 파괴, 조골세포의 손실, 석회화된 골수의 존재, 괴사 부위에 국한된 조골세포의 오스테오칼신 발현 증가로 인한 

리모델링 증가와 골밀도 감소가 나타났습니다. 

종합하면, 이번 연구는 

대퇴골두 무혈성괴사와 관련된 대사체, 병태생리학적, 형태학적 변화를 처음으로 규명하여 

이 쇠약성 질환에 대한 새로운 마커 및 치료 전략 개발에 대한 통찰력을 제공합니다.

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Introduction

Avascular necrosis of femoral head (AVNFH) is a progressive, multifactorial and challenging clinical problem that is on the rise1, 2, mostly affecting the middle aged male population in the most productive age group of 25–50 years. Clinically, AVNFH is a pathological state with multiple etiologies associated with a reduction in the vascular supply to the subchondral bone of the femoral head. This results in osteocyte death and progressive collapse of the articular surface followed by degenerative arthritis of the hip joint. In majority of patients, non-traumatic AVNFH is either associated with use of alcohol3, glucocorticoids4 presence of hematologic disorders (for example: sickle cell anemia5, thalassemia6, polycythemia7, hemophilia8, myeloproliferative disorder9, metabolic disorders (Gaucher disease)10 as well as conditions such as hypercholesterolemia11, pregnancy12, chronic renal failure13, hyperparathyroidism14, Cushing’s disease15 etc. In about 30% of patients however, etiology of non-traumatic AVNFH is unclear and hence these are termed Idiopathic16. The fact that AVNFH is sometimes seen in twins and in familial clusters suggests that genetic factors may be involved17.

Limited insights into the association of AVNFH with coagulation defects as well as lack of suitable animal models18 has resulted in limited knowledge on the pathogenesis of AVNFH. This in turn has hampered the development of predictive markers for disease initiation or progression. Currently, non-invasive diagnostic tests to detect AVNFH include plain radiography, magnetic resonance imaging (MRI), computer assisted tomography (CT), skeletal scintigraphy, and single photon emission computed tomography (SPECT). X-ray based radiographic detection are not sensitive enough to detect AVNFH at its onset (stages 0 and 1) while its overall sensitivity for early stage AVNFH is only about 41%19.

Various causal factors have been described to be associated with the manifestation of this disease. These include genetic (SNP), environmental, alcohol and steroid abuse, infection20 as well as intravascular coagulation21. Among the genetic factors, the most well characterized one describes mutations in Methylenetetrahydrofolate reductase (MTHFR)22, which is seen in certain populations. MTHFR mutations can impact one-carbon metabolism and alter levels of homocysteine (HC). Alternately, HC can also be altered by change in levels of folate or co-factors like vitamins B12 and B623. Elevated levels of HC has been shown to adversely affect bone strength24, while increased plasma concentration of the metabolite are correlated with increased risk for fractures25, Furthermore higher levels of HC and its associated metabolite S-adenosylhomocysteine (SAH) have been reported to cause structural alterations in the bone characterized by increased trabecular separation as well as reduced trabecular thickness, number and area26.

Intriguingly enough, in the context of AVNFH, studies looking at alterations in HC metabolism as well as delineating ultrastructural changes in the affected bone are limited. To fill in this knowledge gap, we have used a combination of clinical data mining, mass spectrometry, histopathology, immunohistochemistry and biophysical techniques like Micro-Raman spectroscopy, Raman Mapping, Computer assisted tomography scanning analysis and Scanning Electron Microscopy (SEM) to delineate key changes associated with AVNFH in patients.

소개

대퇴골두 무혈성 괴사(AVNFH)는

진행성, 다인성, 도전적인 임상 문제로,

주로 25-50세의 가장 생산적인 연령대의 중년 남성 인구에 영향을 미치며1, 2

증가 추세에 있습니다.

임상적으로 AVNFH는

대퇴골두 연골하 뼈로의 혈관 공급 감소와 관련된 다양한 원인을 가진 병리학적인 상태입니다.

이로 인해

골세포가 사멸하고 관절 표면이 점진적으로 붕괴되어 고

관절의 퇴행성 관절염이 발생합니다.

대부분의 환자에서 비외상성 AVNFH 대퇴골두 무혈성괴사 는

알코올3, 글루코코르티코이드4

혈액 질환(예: 겸상 적혈구 빈혈5,

지중해 빈혈6,

적혈구 증식 장애7, 혈우병8, 골수 증식 장애9,

대사 장애(고셔병)10,

고 콜레스테롤 혈증11,

임신12,

만성 신부전13,

부갑상선 기능 항진증14,

쿠싱병15 등과 같은 조건과 연관되어 있습니다.

그러나

약 30%의 환자에서는

비외상성 대퇴골두 무혈성괴사의 원인이 불분명하므로 이를 특발성16 이라고 합니다.

AVNFH가

때때로 쌍둥이와 가족 집단에서 나타난다는 사실은

유전적 요인이 관여할 수 있음을 시사합니다17.

적절한 동물 모델18의 부재뿐만 아니라

응고 결함과의 연관성에 대한 제한된 통찰력으로 인해

AVNFH의 발병 기전에 대한 지식이 제한적이었습니다.

이는 결국 질병의 발병 또는 진행에 대한 예측 마커의 개발을 방해했습니다.

현재 대퇴골두 무혈성괴사를 검출하기 위한 비침습적 진단 검사로는

일반 방사선 촬영,

자기공명영상(MRI),

컴퓨터 보조 단층촬영(CT),

골격 신티그래피,

단일광자방출 컴퓨터 단층촬영(SPECT) 등이 있습니다.

X-선 기반 방사선 검사는

발병 초기(0기 및 1기)에 AVNFH를 감지할 만큼 민감하지 않으며,

초기 AVNFH의 전체 민감도는 약 41%에 불과합니다19.

이 질병의 발현과 관련된 다양한 인과적 요인이 설명되어 있습니다.

여기에는

유전적(SNP), 환경적, 알코올 및 스테로이드 남용, 감염20 및 혈관 내 응고21 등이 포함됩니다.

유전적 요인 중 가장 잘 알려진 것은

특정 집단에서 나타나는

메틸렌테트라하이드로폴레이트 환원효소(MTHFR)22 의 돌연변이를 설명하는 것입니다.

MTHFR 돌연변이는

일탄소 대사에 영향을 미치고

호모시스테인(HC) 수치를 변화시킬 수 있습니다.

또는

엽산이나 비타민 B12 및 B623 과 같은

보조 인자의 수치 변화에 의해서도

HC(호모시스테인)가 변경될 수 있습니다.

HC 수치가 높아지면

뼈 강도에 부정적인 영향을 미치는 것으로 나타났으며24,

대사 산물의 혈장 농도 증가는 골절 위험 증가와 관련이 있습니다25,

또한

HC 및 관련 대사 산물인 S-아데노실호모시스테인(SAH) 수치가 높으면

골절의 두께, 수 및 면적 감소뿐만 아니라

골절 분리 증가를 특징으로 하는

뼈의 구조적 변화를 일으키는 것으로 보고되었습니다26 .

흥미롭게도

AVNFH의 맥락에서 HC 대사의 변화를 살펴보고

영향을 받은 뼈의 초구조적 변화를 묘사하는 연구는 제한적입니다.

이러한 지식 격차를 메우기 위해 저희는 임상 데이터 마이닝, 질량 분석, 조직 병리학, 면역 조직 화학 및 마이크로 라만 분광법, 라만 매핑, 컴퓨터 보조 단층 촬영 스캔 분석 및 주사 전자 현미경(SEM)과 같은 생물 물리학 기술을 조합하여 환자의 AVNFH와 관련된 주요 변화를 묘사했습니다.

Results

Figure 1 shows the overall workflow of the study. Here we used a combination of clinical data mining, metabolomics, biophysical methods and immunohistochemistry to get deeper insights into biochemical, pathophysiological and morphometric attributes associated with Avascular Necrosis of the Femoral Head (AVNFH). The clinical data mining involved statistical analysis of 7 clinical chemistry parameters for their association with AVNFH using a patient de-identified database consisting of 69 AVNFH and 71 control individuals. Additional insights into altered metabolism and associated metabolite levels were obtained using mass spectrometry analysis of AVNFH and control plasma samples. In parallel, Micro-Raman spectroscopy and Raman mapping were used to compare the chemical composition of AVNFH and control bone samples, while the ultrastructure was studied using histopathology, immunohistochemistry and scanning electron microscopy. In addition, computer assisted tomography scans were used to compare the overall bone mineral density in AVNFH and control bone samples.

결과

그림 1은 연구의 전반적인 워크플로우를 보여줍니다. 여기에서는 임상 데이터 마이닝, 대사체학, 생물물리학적 방법, 면역조직화학을 결합하여 대퇴골두 무혈성 괴사(AVNFH)와 관련된 생화학적, 병리생리학적, 형태학적 특성에 대한 심층적인 통찰력을 얻었습니다. 임상 데이터 마이닝에는 69명의 AVNFH 환자와 71명의 대조군으로 구성된 환자 비식별 데이터베이스를 사용하여 7가지 임상 화학 파라미터를 통계적으로 분석하여 AVNFH와의 연관성을 파악하는 작업이 포함되었습니다. 변화된 신진대사 및 관련 대사물질 수치에 대한 추가 인사이트는 AVNFH 및 대조군 혈장 샘플의 질량 분석 분석을 통해 얻었습니다. 이와 동시에 마이크로 라만 분광법과 라만 매핑을 사용하여 AVNFH와 대조군 뼈 샘플의 화학 성분을 비교하고 조직 병리학, 면역 조직 화학 및 주사 전자 현미경을 사용하여 초구조를 연구했습니다. 또한 컴퓨터 보조 단층 촬영 스캔을 사용하여 AVNFH와 대조군 뼈 샘플의 전체 골밀도를 비교했습니다.

Figure 1

Overview of the comprehensive analysis of AVNFH and control samples. Bone and plasma samples as well as retrospective clinical chemistry data from patients with Avascular Necrosis of Femoral Head (AVNFH) and healthy controls were used in this study. Control individuals visited the hospital for treatment of bone fractures and had no history of AVNFH. Retrospective clinical chemistry data from 69 AVNFH and 71 controls were analysed. In addition, age and gender matched plasma samples collected prior to surgery (AVNFH n = 30) and during routine clinical visit (for controls, n = 31) were used for mass spectrometry-based analysis of selected set of metabolites associated with homocysteine pathway. Bone samples were collected post-surgery (AVNFH, n = 13) or during treatment of fractured bones (controls, n = 7) and used for morphological, histopathological, and biophysical studies. Biophysical studies involved analysis of matrix composition using Micro-Raman spectroscopy and Raman mapping, analysis of bone architecture using Scanning Electron Microscopy (SEM), and analysis of bone mineral density using Computer Assisted Tomography scans as described in the text. Red, Green and yellow arrows/lines indicate elevated, reduced and unchanged levels respectively of assayed parameters in AVNFH versus control samples.

AVNFH 및 대조군 샘플에 대한 종합적인 분석 개요. 이 연구에서는 대퇴골두 무혈성 괴사(AVNFH) 환자와 건강한 대조군의 뼈 및 혈장 샘플과 후향적 임상 화학 데이터를 사용했습니다. 대조군은 골절 치료를 위해 병원을 방문했으며 AVNFH 병력이 없는 사람들이었습니다. 69명의 AVNFH 환자와 71명의 대조군의 후향적 임상 화학 데이터를 분석했습니다. 또한 호모시스테인 경로와 관련된 선택된 대사물질 세트의 질량 분석 기반 분석을 위해 수술 전(AVNFH n = 30)과 일상적인 임상 방문 시(대조군, n = 31) 수집한 연령 및 성별이 일치하는 혈장 샘플을 사용했습니다. 뼈 샘플은 수술 후 또는 골절된 뼈의 치료 중(대조군, n = 13) 수집하여 형태학적, 조직병리학적, 생물물리학적 연구에 사용했습니다(AVNFH, n = 7). 생물물리학적 연구에는 마이크로 라만 분광법 및 라만 매핑을 사용한 매트릭스 구성 분석, 주사 전자 현미경(SEM)을 사용한 뼈 구조 분석, 컴퓨터 보조 단층 촬영 스캔을 사용한 골밀도 분석이 포함되었습니다(본문에서 설명한 대로). 빨간색, 녹색 및 노란색 화살표/선은 각각 대조 샘플과 비교하여 AVNFH에서 분석된 파라미터의 증가, 감소 및 변하지 않은 수준을 나타냅니다.

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Results of these detailed characterization studies revealed elevated plasma levels of metabolites in the methionine-homocysteine pathway, urea pathway and polyamines in AVNFH patients. Further, bone from AVNFH patients showed unique changes in ratio of matrix components which was reflected in ultrastructural alterations describing the loss of trabecular connectivity, presence of micro-cracks, appearance of creeping substitutions, presence of areas containing dead bones and reduced bone density. Taken together, these findings demonstrate extensive bone remodelling in the diseased bone, which is consistent with increased expression‎ of osteocalcin in these samples.

Alterations in specific biochemical parameters are observed in AVNFH

To begin with, we examined the clinical chemistry values for 7 parameters and compared them between AVNFH patients (n = 68, refer (Supplementary Table S5) for clinical features of the AVNFH cohort) and control individuals (n = 71). Control individuals selected for this study had no prior history of AVNFH and presented at Sri Sathya Sai Institute for Higher Medical Sciences (SSSIHMS) India, for treatment of bone fractures. Supplementary Figure 1 shows the average distribution of the levels of clinical analytes that were compared between AVNFH patients and control individuals. Interestingly, as shown in Fig. 2A and C, levels of random glucose and hemoglobin were significantly down-regulated in AVNFH patients compared to controls. In contrast, levels of serum creatinine and sodium were significantly elevated in AVNFH patients (Fig. 2B,D). These findings indicate presence of an altered metabolism in AVNFH patients, which is consistent with reports describing elevated levels of homocysteine AVNFH patients.

이러한 세부 특성화 연구 결과,

AVNFH 환자에서

메티오닌-호모시스테인 경로,

요소 경로 및 폴리아민의 혈장 내 대사산물의 수치가 상승한 것으로 나타났습니다.

또한

AVNFH 환자의 뼈는 매트릭스 성분의 비율에 독특한 변화를 보였는데,

이는 소주골 연결성 상실,

미세 균열의 존재,

크리핑 치환의 출현,

죽은 뼈가 있는 영역의 존재 및 골밀도 감소를 설명하는 초구조적 변화에 반영되어 있었습니다.

이러한 결과를 종합해 보면,

질병에 걸린 뼈에서 광범위한 뼈 리모델링이 일어나고 있으며,

이는 이러한 샘플에서 오스테오칼신의 발현 증가와 일치합니다.

특정 생화학적 파라미터의 변화는 AVNFH에서 관찰됩니다.

우선, 7가지 파라미터에 대한 임상 화학 수치를 조사하여 AVNFH 환자(n = 68, AVNFH 코호트의 임상적 특징은 (보충 표 S5) 참조)와 대조군(n = 71) 간에 비교했습니다. 이 연구를 위해 선정된 대조군은 AVNFH 병력이 없고 인도 스리 사티아 사이 고등 의학 연구소(SSSIHMS)에서 골절 치료를 받은 적이 없는 사람들입니다. 부록 그림 1은 AVNFH 환자와 대조군 간에 비교한 임상 분석 수치의 평균 분포를 보여줍니다.

흥미롭게도 그림 2A와 C에서 볼 수 있듯이,

AVNFH 환자는 대조군에 비해

무작위 포도당과 헤모글로빈 수치가 현저히 낮게 조절되었습니다.

대조적으로,

혈청 크레아티닌과 나트륨 수치는

AVNFH 환자에서 유의하게 상승했습니다(그림 2B,D).

이러한 결과는

AVNFH 환자의 신진대사에 변화가 있음을 나타내며,

이는 호모시스테인 AVNFH 환자의 수치가 상승했다는 보고와 일치합니다.

Figure 2

Box plots showing significantly altered levels of clinical analytes in blood samples from AVNFH patients compared to control subjects. X-axis describes the diagnostic groups and Y-axis represents concentrations of the analyte. (A) Random glucose, (B) Sodium, (C) Hemoglobin, and (D) Serum creatinine. In all cases p values were calculated using Students T-test. One outlier AVNFH sample having significantly lower values for all the clinical parameters was removed from the analysis.

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Targeted plasma metabolomics in AVNFH reveals changes in methionine-homocysteine pathway metabolites

To obtain further insights into altered metabolism and to verify elevated levels of homocysteine in AVNFH, liquid chromatography mass spectrometry (LC-MS) analysis was carried out initially using plasma samples from AVNFH patients (n = 14) and control individuals (n = 14). Specifically, Single Reaction Monitoring (SRM)-based targeted analyses were used to measure the relative levels of metabolites associated with methionine-homocysteine/transsulfuration pathway and polyamine metabolism. Metabolites were extracted using a standardized protocol described under the Methods section. A total of 15 metabolites that included methionine, homocysteine, S-adenosyl methionine (SAM), S-adenosyl homocysteine (SAH), adenosine, betaine, cystathionine, ornithine, arginine, proline, spermine, spermidine and putrescene were measured. In addition, levels of vitamins B6 and B12 that serve as co-factors in the regeneration of methionine from homocysteine were also measured (Refer to Supplementary Table S1 for list of metabolites and their associated SRM transitions).

Importantly, in our initial analysis, levels of methionine, SAM, SAH, homocysteine and adenosine were significantly (P<0.01) elevated in plasma of AVNFH patients compared to age-matched controls (Fig. 3A-E). These findings are consistent with earlier reports that implicate a role for homocysteine in AVNFH27. In addition, consistent with elevated SAM levels as well as in line with our earlier observation on elevated levels of circulating creatinine in AVNFH (Fig. 2D), AVNFH plasma had significantly elevated levels of polyamines namely spermine and spermidine (Fig. 3I).

메티오닌-호모시스테인 경로 대사물질의 변화를 밝혀낸 AVNFH의 표적 혈장 대사체학

변화된 대사에 대한 추가 통찰력을 얻고

AVNFH에서 호모시스테인 수치의 상승을 확인하기 위해

먼저 AVNFH 환자(n = 14)와 대조군(n = 14)의 혈장 샘플을 사용하여

액체 크로마토그래피 질량 분석법(LC-MS)을 수행했습니다.

특히 단일 반응 모니터링(SRM) 기반 표적 분석을 사용하여 메티오닌-호모시스테인/환황화 경로 및 폴리아민 대사와 관련된 대사물질의 상대적 수준을 측정했습니다. 대사산물은 방법 섹션에 설명된 표준화된 프로토콜을 사용하여 추출했습니다.

메티오닌, 호모시스테인, S-아데노실 메티오닌(SAM), S-아데노실 호모시스테인(SAH), 아데노신, 베타인, 시스타티오닌, 오르니틴, 아르기닌, 프롤린, 스페르민, 스페르미딘 및 푸트레센을 포함한 총 15가지 대사산물이 측정되었습니다.

또한

호모시스테인에서 메티오닌을 재생하는 데 보조 인자로 작용하는

비타민 B6와 B12의 수준도 측정했습니다

(대사산물 및 관련 SRM 전이 목록은 보충 표 S1 참조).

중요한 것은 초기 분석에서 연령이 일치하는 대조군에 비해

AVNFH 환자의 혈장에서

메티오닌, SAM, SAH, 호모시스테인 및 아데노신 수치가

유의하게(P<0.01) 상승했다는 점입니다(그림 3A-E).

이러한 결과는 AVNFH에서 호모시스테인의 역할을 암시하는 이전 보고와 일치합니다27. 또한, 상승된 SAM 수치와 일치할 뿐만 아니라 AVNFH의 순환 크레아티닌 수치 상승에 대한 이전의 관찰과 일치합니다(그림 2D), AVNFH 혈장은 폴리아민, 즉 스페르민과 스페르미딘의 수치가 유의하게 높았습니다(그림 3I).

Figure 3

Overview showing normalized levels of selected metabolites in plasma of AVNFH patients (n = 14) and control (n = 14) individuals. Levels of spiked internal standard (see text) were used to normalize the data. P values were computed using Students T-test between the AVNFH (black bars) and control (open bars) groups. Overall levels of metabolites in the folate-methionine Pathway (A) Methionine, (B) S- adenosyl methionine (SAM), (C) S-adenosyl Homocysteine (SAH), (D) Homocysteine, and (E) Adenosine, were significantly elevated in AVNFH patients. Further levels of (F) Betaine, as well as cofactors vitamins (G) B12 and (H) B6 were significantly reduced in AVNFH samples compared to controls. Also, levels of polyamines namely (I) Spermine and Spermidine were significantly elevated in AVNFH samples compared to controls. P values for each comparison are included in each figure panel.

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Accumulation of homocysteine in AVNFH patients is further supported by decreased levels of betaine and reduced levels of vitamins B12 and B6, both of which serve as co-factors in synthesis of methionine from homocysteine (Fig. 3F-H). Taken together, these findings suggest that reconversion of homocysteine to methionine is significantly hampered in AVNFH.

To verify this premise, we carried out a correlation between homocysteine and vitamins B6/B12 using raw spectral intensity data. Furthermore, we also measured these metabolites in an additional set of plasma samples from AVNFH patients (n = 16) and healthy controls (n = 17), in two independent experiments. Importantly, in each of the three independent experiments, Pearson Correlation analysis revealed a strong negative correlation between homocysteine and vitamins B6 (Pearson Correlation range: −0.4 to −0.8, P≤0.05); and between homocysteine and vitamin B12 (Pearson Correlation range: −0.5 to −0.8, P<0.05, Supplementary Table S3). In addition, a similar analysis revealed a strong positive correlation between levels of homocysteine and polyamines that included both spermine (Pearson Correlation range: 0.4 to 0.6, P<0.05) and spermidine (Pearson Correlation range: 0.4 to 0.7, P<0.05).

Micro-Raman spectroscopic analysis of AVNFH Bone shows significant changes in mineral-matrix and chemical composition

Having observed significant changes in the biochemical and metabolic profile in blood and plasma of AVNFH patients, we then asked if any of these alterations could be associated with changes in the bone architecture and composition. Figure 4A and B, and Supplementary Figure 2, shows representative cross sections of surgically resected trabecular bones from control individuals (having fractured femur) and AVNFH patients. AVNFH diagnosis was confirmed using X-ray images of the pelvic region (Fig. 4C,D and Supplementary Figure 3) and/or Magnetic Resonance Imaging (MRI, Fig. 4E,F and Supplementary Figure 4). X-ray images of AVNFH patients revealed distinct collapse of the subchondral bone with associated sclerotic changes (Fig. 4C,D and Supplementary Figure 3). Consistent with this, Magnetic Resonance Imaging (MRI) of AVNFH patients shows regions of avascular necrosis in the trabeculae of the femoral bone (Fig. 4E,F and Supplementary Figure 4).

호모시스테인에서 메티오닌을 합성하는 보조 인자로 작용하는

베타인 수치가 감소하고

비타민 B12와 B6 수치가 감소하는 것으로도

AVNFH 환자의 호모시스테인 축적을 뒷받침합니다(그림 3F-H).

이러한 결과를 종합해 볼 때,

호모시스테인이 메티오닌으로 재전환되는 것이

AVNFH에서 상당히 방해받고 있음을 시사합니다.

이 전제를 검증하기 위해

원시 스펙트럼 강도 데이터를 사용하여

호모시스테인과 비타민 B6/B12 간의 상관관계를 조사했습니다.

또한 두 개의 독립적인 실험에서

AVNFH 환자(n = 16)와 건강한 대조군(n = 17)의 추가 혈장 샘플 세트에서 이러한 대사산물을 측정했습니다.

중요한 것은

세 가지 독립 실험 각각에서 피어슨 상관관계 분석 결과

호모시스테인과 비타민 B6(피어슨 상관관계 범위: -0.4 ~ -0.8, P≤0.05),

호모시스테인과 비타민 B12(피어슨 상관관계 범위: -0.5 ~ -0.8, P<0.05, 보충 표 S3) 사이에

강한 음의 상관관계가 있다는 사실이 밝혀졌다는 점입니다.

또한 유사한 분석 결과,

호모시스테인과 폴리아민 수치 사이에는

스페르민(피어슨 상관관계 범위: 0.4~0.6, P<0.05)과 스페르미딘(피어슨 상관관계 범위: 0.4~0.7, P<0.05) 모두

강한 양의 상관관계가 있는 것으로 나타났습니다.

AVNFH 뼈의 마이크로 라만 분광 분석은 미네랄 매트릭스 및 화학적 구성에 상당한 변화를 보여줍니다.

AVNFH 환자의 혈액과 혈장에서 생화학 및 대사 프로필의 유의미한 변화를 관찰한 후, 이러한 변화가 뼈 구조 및 구성의 변화와 관련이 있는지 여부를 조사했습니다. 그림 4A와 B, 보충그림 2는 대조군(대퇴골 골절이 있는) 및 AVNFH 환자의 수술로 절제된 소주골의 대표적인 단면을 보여줍니다. 골반 부위의 X-레이 이미지(그림 4C,D 및 보충 그림 3) 및/또는 자기공명영상(MRI, 그림 4E,F 및 보충그림 4)을 사용하여 AVNFH 진단을 확인했습니다. AVNFH 환자의 X-레이 이미지에서 연골하 뼈의 뚜렷한 붕괴와 관련 경화성 변화가 발견되었습니다(그림 4C,D 및 보충 그림 3). 이와 일치하는 AVNFH 환자의 자기공명영상(MRI)은 대퇴골 소주골의 무혈성 괴사 부위를 보여줍니다(그림 4E,F 및 보충 그림 4).

Figure 4

(A) Overview of representative AVNFH and control bone samples used in the study. (A) A representative gross image of a trabecular cross section of control bone. (B) Same as in A, but for AVNFH bone. Region affected by AVN is shown by the yellow arrow. (C) X ray image of the pelvic region from a control individual with fractured femur (D) X ray image of the pelvic region of a patient with Avascular Necrosis of the Femoral Head (AVNFH). (E,F) Magnetic Resonance Image (MRI of the pelvic region of a patient with AVNFH.

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To obtain insights into the chemical composition of the bone, we used various biophysical methods to examine the trabecular cross sections (4–5 cm in diameter, refer Supplementary Figure 2) obtained from 6 AVNFH patients and 3 control individuals. When examined under a stereo microscope, the trabecular cross section of the control bone was found to be homogenous without any gross surface alterations. In contrast, cross sections of AVNFH bones contained both the necrotic avascular region and adjacent non-diseased areas. For all the AVNFH bone samples except one (Supplementary Figure 2F), necrotic regions were grossly delineated from adjacent non-disease areas by the clinician post-resection (Fig. 4B, Yellow arrows).

Initially, we carried out Micro-Raman spectroscopy analysis of these trabecular sections. To minimize artefacts in Raman spectral analysis, bone samples were examined without prior chemical treatment. Supplementary Figure 5A shows a representative Raman spectrum for a healthy bone with sequentially labelled peaks that define distinct chemical components listed in Supplementary Table S2. Thus for example, the most prominent peak labelled peak 11 at 1002 cm−1 describes the phenylalanine content of the bone while peaks 960 cm−1, 1450 cm−1 and 1660 cm−1 describe the phosphate, collagen and amide content respectively (Supplementary Figure. 5A). Using this annotation, we analysed the Raman spectra obtained from AVNFH and control bones.

Raman spectra were collected across the cross section of the trabecular bone at multiple pre-identified spots (n~16) that were 2600–3000 microns apart. For example, as shown for a representative AVNFH bone (Supplementary Figure 5B), total of 17 spectra were collected, of which 11 were in non-diseased areas (green spots, S1–S6 and S7–S11) and 6 were within the necrotic region (red spots, S1-S6). Importantly, the Raman spectra obtained for control and AVN samples were comparable to the reference spectra shown in Supplementary Figure 5A with well resolved bands for phosphate (960 cm−1 and 430 cm−1), carbonate (1070 cm−1), collagen (1446 cm−1 and 1568 cm−1) and phenylalanine (1002 cm−1).

To begin with, for each sample, all the peak intensities within each spectra was normalized using the corresponding intensity for the phenylalanine peak28. We then compared the intensity of the phosphate, carbonate and amide peaks obtained from the control bones with the corresponding intensities obtained from non-diseased and necrotic areas in the AVNFH bone. Following this, we calculated the mineral to matrix ratio (phosphate to amide ratio), carbonate to phosphate ratio, carbonate to amide ratio and the half width of phosphate peak (at 960 cm−1), for samples in each of the three groups.

Overall, the control bone had significantly higher values for phosphate to amide ratio and carbonate to amide ratio compared to adjacent non-diseased and necrotic regions (AVNFH, Fig. 5A top and middle panel P<0.05). These ratios were comparable between necrotic and the non-diseased adjacent regions (Fig. 5A). On the other hand, the carbonate to phosphate ratio remained unchanged for all the comparisons (Supplementary Figure 6A). The extent of crystallinity defined by half width of phosphate band (960 cm−1) showed a modest reduction in AVNFH regions compared to control (P<0.08, Supplementary Figure 6B). Furthermore, the normalized intensity of only the carbonate peak (1070 cm−1) was found to be significantly reduced in necrotic sites compared to control (P<0.01, Fig. 3A, bottom panel).

뼈의 화학적 구성에 대한 통찰력을 얻기 위해 다양한 생물물리학적 방법을 사용하여 6명의 AVNFH 환자와 3명의 대조군에게서 얻은 소주골 단면(직경 4~5cm, 보충 그림 2 참조)을 검사했습니다. 실체현미경으로 검사한 결과, 대조군 뼈의 소주골 단면은 표면의 심한 변화 없이 균질한 것으로 나타났습니다. 이와 대조적으로 AVNFH 뼈의 단면은 괴사된 무혈관 영역과 인접한 비질환 영역이 모두 포함되어 있었습니다. 하나를 제외한 모든 AVNFH 뼈 샘플 (보충 그림 2F)의 경우, 임상의가 절제 후 인접한 비질환 영역에서 괴사 영역이 심하게 구분되었습니다(그림 4B, 노란색 화살표).

처음에는 이러한 섬유주 섹션에 대해 마이크로 라만 분광 분석을 수행했습니다. 라만 스펙트럼 분석에서 인공물을 최소화하기 위해 뼈 샘플은 사전 화학 처리 없이 검사했습니다. 보충 그림 5A는 건강한 뼈의 대표적인 라만 스펙트럼으로, 보충 표 S2 에 나열된 고유한 화학 성분을 정의하는 피크가 순차적으로 표시되어 있습니다. 예를 들어, 가장 두드러진 피크인 1002 cm-1의 피크 11은 뼈의 페닐알라닌 함량을 나타내며 피크 960 cm-1, 1450 cm-1 및 1660 cm-1은 각각 인산염, 콜라겐 및 아미드 함량을 나타냅니다(보충 그림 5A). 이 주석을 사용하여 AVNFH와 대조군 뼈에서 얻은 라만 스펙트럼을 분석했습니다.

라만 스펙트럼은 2600-3000미크론 간격으로 미리 식별된 여러 지점(n~16)에서 소주골의 단면을 가로질러 수집되었습니다. 예를 들어, 대표적인 AVNFH 뼈(보충 그림 5B)에 표시된 것처럼 총 17개의 스펙트럼이 수집되었으며, 이 중 11개는 비질환 영역(녹색 점, S1-S6 및 S7-S11)에, 6개는 괴사 영역(빨간색 점, S1-S6) 내에 있었습니다. 중요한 것은 대조군 및 AVN 샘플에서 얻은 라만 스펙트럼이 인산염(960cm-1 및 430cm-1), 탄산염(1070cm-1), 콜라겐(1446cm-1 및 1568cm-1), 페닐알라닌(1002cm-1)에 대해 잘 분해된 밴드로 보충그림 5A에 나타난 기준 스펙트럼과 비슷하다는 점입니다.

우선, 각 샘플에 대해 각 스펙트럼 내의 모든 피크 강도를 페닐알라닌 피크에 해당하는 강도를 사용하여 정규화했습니다28. 그런 다음 대조군 뼈에서 얻은 인산염, 탄산염 및 아미드 피크의 강도를 AVNFH 뼈의 비질환 및 괴사 부위에서 얻은 해당 강도와 비교했습니다. 그 후 세 그룹의 각 샘플에 대해 미네랄 대 매트릭스 비율(인산염 대 아미드 비율), 탄산염 대 인산염 비율, 탄산염 대 아미드 비율 및 인산염 피크의 절반 폭(960cm-1에서)을 계산했습니다.

전반적으로 대조군 뼈는 인접한 비질환 및 괴사 부위에 비해 인산염 대 아미드 비율과 탄산염 대 아미드 비율이 유의하게 높았습니다(AVNFH, 그림 5A 상단 및 중간 패널 P<0.05). 이러한 비율은 괴사 부위와 비병변 인접 부위 간에 비슷했습니다(그림 5A). 반면에 탄산염 대 인산염 비율은 모든 비교에서 변하지 않았습니다(보충 그림 6A). 인산염 밴드의 절반 폭(960 cm-1)으로 정의된 결정성의 정도는 대조군에 비해 AVNFH 영역에서 완만하게 감소한 것으로 나타났습니다(P<0.08, 보충 그림 6B). 또한 탄산염 피크(1070 cm-1)만의 정규화된 강도는 대조군에 비해 괴사 부위에서 유의하게 감소한 것으로 나타났습니다(P<0.01, 그림 3A, 하단 패널).

Figure 5

Micro- Raman spectroscopy and computer assisted tomography analysis of AVNFH and control bone. (A) Box plot showing the ratio of phosphate to amide peaks (top panel), carbonate to amide peak (middle panel), and carbonate peak intensity, in control, adjacent non-diseased and AVNFH bone samples obtained using micro-Raman line scan analysis. All the three parameters were significantly reduced in necrotic regions compared to control bone. The carbonate peak intensity was not significantly different between control and non-diseased adjacent areas. (B) Photomicrographs obtained from Raman mapping analysis showing the distribution of carbonate and phosphate in control, adjacent non-diseased and necrotic regions. Shades of red indicate elevated levels and shades of blue indicate reduced levels. Shades of green indicate intermediate levels (refer to color scale). (C) same as in B, but for phosphate and amide peaks, (D) box plot showing quantitation of D. Median values derived from one hundred data points is shown. Carbonate to phosphate ratio progressively increased from control to adjacent non-diseased to necrotic areas, (E) same as in D, but for phosphate to amide ratio. Phosphate to amide ratio progressively decreased from control to adjacent non-diseased to necrotic areas, (F) box plot showing the comparison of median CT intensities quantified by Hounsfield unit values (HU) for Control (n = 4) and AVNFH (n = 6) bone samples. Median HU value that denotes the radio density on a CT scan was significantly lower in AVNFH patients compared to control individuals. Radio density is a surrogate measure for bone mineral density. All P values described in the figure panels were calculated using Student T-tests.

AVNFH 및 대조군 뼈의 마이크로 라만 분광법 및 컴퓨터 보조 단층촬영 분석.

(A) 마이크로 라만 라인 스캔 분석을 사용하여 얻은 대조군, 인접 비질환 뼈 샘플 및 AVNFH 뼈 샘플에서 인산염 대 아미드 피크(상단 패널), 탄산염 대 아미드 피크(중간 패널) 및 탄산염 피크 강도의 비율을 보여주는 박스 플롯. 세 가지 파라미터 모두 대조군 뼈에 비해 괴사 부위에서 유의미하게 감소했습니다. 탄산염 피크 강도는 대조군과 비질환 인접 부위 간에 큰 차이가 없었습니다.

(B) 라만 매핑 분석에서 얻은 현미경 사진으로 대조군, 인접한 비질환 및 괴사 영역에서 탄산염과 인산염의 분포를 보여줍니다. 빨간색 음영은 증가된 수준을 나타내고 파란색 음영은 감소된 수준을 나타냅니다. 녹색 음영은 중간 수준을 나타냅니다(색 눈금 참조).

(C)는 B와 동일하지만 인산염 및 아미드 피크의 경우,

(D)는 100개의 데이터 포인트에서 도출된 D의 정량을 나타내는 박스형 플롯입니다. 탄산염 대 인산염 비율은 대조군에서 인접한 비질환-괴사 영역으로 갈수록 점진적으로 증가하며,

(E)는 D와 동일하지만 인산염 대 아미드 비율의 경우입니다. 인산염 대 아미드 비율은 대조군에서 인접한 비질환-괴사 영역으로 갈수록 점진적으로 감소했습니다.

(F) 대조군(n = 4) 및 AVNFH(n = 6) 뼈 샘플의 하운스필드 단위 값(HU)으로 정량화된 CT 강도의 중앙값 비교를 보여주는 박스 플롯입니다. CT 스캔에서 방사선 밀도를 나타내는 HU 값의 중앙값은 대조군에 비해 AVNFH 환자에서 유의하게 낮았습니다. 방사선 밀도는 골밀도의 대리 척도입니다. 그림 패널에 설명된 모든 P값은 학생 T-검정을 사용하여 계산되었습니다.

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To further confirm‎ the AVNFH associated changes in chemical composition identified by the Raman line scans, we carried out Raman Mapping studies on an independent group of 6 AVNFH and 4 control bone samples. A total of 100 Raman spectra were individually collected from the adjacent non-diseased and necrotic regions in AVNFH bones as well as from the normal areas in the control bone samples. Interestingly, in contrast to the results of the gross Raman analysis described above, these fine mapping studies revealed significantly higher carbonate: phosphate ratio in AVNFH bone compared to healthy control bone (P = 0.01, Fig. 5B and Supplementary Figure 7). Further, within the AVNFH bone, the carbonate:phosphate ratio showed a progressive increase from adjacent non-diseased region to the AVN region (Fig. 5D and Supplementary Figure 7). In contrast however, the mineral:matrix (959/1660 cm−1) ratio was significantly reduced in AVNFH bone compared to control bone (P = 0.0005, Fig. 5C and E and Supplementary Figure 8). Also, within the AVNFH bone, the mineral:matrix ratio showed a progressive decrease from adjacent non-diseased region to the AVN region (Fig. 5E). Taken together, these studies for the first time confirm‎ alterations in the chemical and matrix composition of the AVNFH bone.

Analysis of computer assisted tomography Scans reveals reduced bone mineral density in AVNFH bone

To determine whether the changes in chemical composition could influence bone mineral density, we obtained radio density of the bone samples that were earlier analysed by Raman mapping. Radio density, used as a surrogate for bone mineral density, was calculated from the computer assisted tomography (CT) scans and represented as standardized Hounsfield Units (HU, Fig. 5F, Supplementary Figures 9–20). Interestingly, 6/6 AVNFH trabecular bone slices that had earlier shown high carbonate:phosphate ratio and low mineral:matrix ratio in the Raman analysis, also had significantly lower median HU values compared to controls (n = 4) (Fig. 5E). 1/6 AVNFH trabecular bone sample was an outlier with high HU values comparable to the control (Supplemental Figure 20). This outlier effect could be potentially attributed to the presence of subchondral sclerotic areas in the AVNFH sample. Taken together, all these findings suggest significant changes in the ultrastructure of the AVNFH bone compare to control bone.

라만 라인 스캔으로 확인된 AVNFH와 관련된 화학 성분의 변화를 추가로 확인하기 위해 6개의 AVNFH 및 4개의 대조군 뼈 샘플을 대상으로 라만 매핑 연구를 수행했습니다. 총 100개의 라만 스펙트럼을 대조군 뼈 샘플의 정상 영역뿐만 아니라 AVNFH 뼈의 인접한 비질환 및 괴사 영역에서 개별적으로 수집했습니다.

흥미롭게도, 위에서 설명한 총 라만 분석 결과와는 대조적으로,

이러한 정밀 매핑 연구에서는

건강한 대조군 뼈에 비해 AVNFH 뼈의 탄산염: 인산염 비율이 상당히 높은 것으로 나타났습니다

(P = 0.01, 그림 5B 및 보충 그림 7).

또한

AVNFH 뼈 내에서 탄산염:인산염 비율은 인접한 비질환 영역에서

AVN 영역으로 갈수록 점진적으로 증가하는 것으로 나타났습니다(그림 5D 및 보충 그림 7).

그러나

대조적으로, 미네랄:매트릭스(959/1660 cm-1) 비율은 대조군 뼈에 비해 AVNFH 뼈에서 유의하게 감소했습니다(P = 0.0005, 그림 5C 및 E와 보충 그림 8). 또한 AVNFH 뼈 내에서 미네랄:매트릭스 비율은 인접한 비질환 영역에서 AVN 영역으로 갈수록 점진적으로 감소하는 것으로 나타났습니다(그림 5E). 이러한 연구 결과를 종합하면, AVNFH 뼈의 화학적 및 매트릭스 구성의 변화가 처음으로 확인되었습니다.

컴퓨터 보조 단층 촬영 스캔을 분석한 결과 AVNFH 뼈의 골밀도가 감소한 것으로 나타났습니다.

화학적 구성의 변화가 골밀도에 영향을 미칠 수 있는지 확인하기 위해 앞서 라만 매핑으로 분석했던 뼈 샘플의 방사능 밀도를 얻었습니다. 골밀도의 대용물로 사용되는 방사선 밀도는 컴퓨터 보조 단층 촬영(CT) 스캔에서 계산되어 표준화된 하운스필드 단위(HU, 그림 5F, 보충 그림 9-20)로 표시되었습니다. 흥미롭게도 라만 분석에서 탄산염:인산염 비율이 높고 미네랄:매트릭스 비율이 낮았던 6/6 AVNFH 골편은 대조군(n = 4)에 비해 HU 중앙값이 현저히 낮았습니다(그림 5E). 1/6 AVNFH 소주골 샘플은 대조군과 비슷한 높은 HU 값을 가진 이상치였습니다(보충 그림 20). 이 이상값 효과는 잠재적으로 AVNFH 샘플에 연골하 경화성 영역이 존재하기 때문일 수 있습니다. 이러한 모든 결과를 종합하면, 대조군 뼈에 비해 AVNFH 뼈의 초구조에 상당한 변화가 있음을 시사합니다.

Scanning Electron Microscopy reveals changes in ultra-structure of AVNFH bone

In order to study the changes in ultra-structure of AVNFH bone samples, Scanning Electron Microscopy (SEM) was carried out on bone slices, at 30–50 X and 5000x magnifications with minimal pre-treatment of the bone samples. Overall, both control and AVNFH bones exhibited the prototypic architecture (Fig. 6A –F) characterized by the presence of multiple interconnected trabeculae forming honey comb like structures. Further, all the bones displayed resting, resorption and formative surfaces. In the control bone and more prominently in the AVNFH bones, trabeculae were broken with associated loss of connective bridges (Fig. 6A –F).

Figure 6

Representative Scanning Electron Microscopic (SEM) images of control and AVNFH bone. (A–C) SEM images of control bone showing honey comb shaped trabecular arrangement. Regions of bone remodelling is also observed (red arrow). (D–F) SEM images of AVNFH bone showing perturbed trabecular arrangement. Trabeculae lose connectivity altering the honey comb shape. In addition, regions of dead bones are observed. (Black areas indicated by red arrow). Extensive remodelling of the bone is indicated by red arrows.

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Interestingly, in the AVNFH bones both the resorption and formative surfaces were predominantly more evident and more extensive compared to controls (Fig. 6D,E and F). Furthermore, AVNFH bones were characterized by the presence of unique dark regions representing areas of bone loss (Fig. 6E,F). These bones also displayed higher surface charging during imaging (data not shown) and calcified marrow regions containing micro-cracks (Fig. 6F). The presence of areas of bone loss (dead bones) was a characteristic feature seen only in AVNFH samples. These findings suggested the existence of extensive remodelling in AVNFH bone compared to the control bone. High power examination of these regions having bone remodelling in both AVNFH and control bones displayed disrupted arrangement of lamellar plates containing disorganized collagen bundles that had lost their polarity (Supplementary Figures 21A,B for AVNFH bone and 21C for control bone).

Osteocalcin Staining reveals active bone remodelling in AVNFH

To further verify the presence of extensive remodelling in AVNFH bones compared to control bone samples, we examined the expression‎ of osteocalcin, a marker for bone remodelling. Immunohistochemistry for osteocalcin revealed significantly higher osteoblast staining in AVNFH bone samples compared to control bone samples (Fig. 7A-C, Supplementary Figure 22). Interestingly, within the AVNFH bone, regions distal to the diseased site showed significantly lesser osteoblast staining compared to regions that were proximal to the affected area (Fig. 7D and E). Furthermore, osteoblasts associated with early fracture callus in the control bones showed significantly lower staining for osteocalcin (Supplementary Figure 22F and H). In contrast, in one of the control bone samples containing late fracture callus, strong osteocalcin staining was observed in osteoblasts (Supplementary Figure 22G). Unlike its expression‎ in the bone tissue, osteocalcin levels in plasma were not significantly different between the control and AVNFH patients (Supplementary Figure 23). Also, levels of serum parathyroid hormone (PTH), an independent bone resorption marker, were also unchanged between AVNFH patients and control individuals (Supplementary Figure 24). Additionally, levels of CTX (Beta-Crosslaps), an independent bone resorption marker was eval‎uated in a new independent cohort of 9 AVNFH patients and 9 healthy control individuals. The results are summarized in Supplementary Table S7A and S7B. Normal diagnostic reference was available only for individuals above the age of 30 years, in the clinical laboratory where CTX assays were carried out. Given this, for individuals below the age of 30 years, reference ranges from three independent diagnostic laboratories namely Mayo clinic29, Quest Diagnostics30 and Arup lab31 were used to interpret the data. Importantly, in the age group above 30 years, CTX values exceeded the diagnostic threshold in 3/7 AVN patients (~43%) and only in 1/7 (~14%) controls (Supplementary Table S7A). Along the same lines, for 2 AVN and 3 controls below the age of 30 years, only 1/2 AVN had their CTX values beyond the reference threshold (Supplementary Table S7B). Overall, using this small group of AVN patients and controls, it appears that higher CTX values are more pronounced in AVN patients compared to controls.

Figure 7

Photomicrographs showing osteocalcin staining in trabecular bone sections from AVNFH patients (panels A–G) and control individuals (panels H-K). Osteocalcin staining in osteoblasts lining (red arrows) the bone were quantified (panel L) by an expert bone pathologist. Overall, strong osteocalcin staining was observed in osteoblasts lining the necrotic areas in AVNFH sample, indicating extensive bone remodelling (panel L). In contrast, osteoblasts lining the control bone showed weak osteocalcin staining (panel L). Interestingly, within the AVNFH osteocalcin (panel L, refer Supplementary Figure 22). In control bones (derived from sites of fracture), osteoblasts proximal to late stage fracture callus showed strong osteocalcin staining, also suggesting extensive bone remodelling (refer Supplementary Figure 22). In control bones (derived from sites of fracture), osteoblasts proximal to late stage fracture callus showed strong osteocalcin staining, also suggesting extensive bone remodelling (refer Supplementary Figure 22). In all cases, non-specific staining was observed in the marrow. All images photographed at 200X (refer to scale in the Inset).

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Histopathology of AVNFH bone verifies structural alterations in the trabeculae

Histopathology of control bones displayed normal trabecular architecture and marrow devoid of necrosis (Fig. 8A). In contrast, AVNFH bone contained a number of reversal cement lines, both in the necrotic as well as in the adjacent non-diseased region, indicative of extensive remodelling (Fig. 8B-F). This is consistent with the SEM and osteocalcin staining data wherein both formative and resorption areas were predominant in AVNFH. Furthermore, the AVNFH bone sections also reveal a number of empty lacunae resulting from the loss of osteocytes as well as poorly vascularised calcified marrow (Fig. 8E,F).

Figure 8

Hematoxylin and Eosin (H&E) - based histopathological characterization of AVNFH and control bone. (A) Section of a typical control bone, (B) section of a trauma induced AVNFH sample showing a large number of cement lines in the trabeculae reflecting sites of excessive bone-remodelling (black arrow), (C) section of non-adjacent diseased region in an AVNFH bone showing large number of empty lacunae in the trabaculae (black arrow), (D) section of AVNFH bone showing necrosis of the marrow associated with affected area (black arrow), (E) section of AVNFH bone showing the presence of calcified marrow associated with adjacent non-diseased areas of the bone (black arrow), (F) section of AVNFH bone showing the presence of poorly vascularized fibrous marrow in the necrotic areas (black arrow).

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Discussion

In this study we have used a multi-pronged approach to characterize AVNFH. A couple of earlier studies have described AVNFH to be a metabolic disorder32, 33. These studies describe altered lipid metabolism and altered liver function to correlate with AVNFH32, 33 as well as implicated a role for homocysteine in this disease27. However, there are no studies to date that examine these metabolic alterations or characterize the levels of homocysteine in AVNFH.

In this study, the initial evidence supporting altered biochemistry in AVNFH was obtained from the retrospective and prospective clinical chemistry data. Interestingly, our analysis revealed significantly lower levels of hemoglobin (Hb) in AVNFH patients. Previous reports have described decreased levels of Hb in AVNFH patients specifically in the context of co-occurrence of sickle cell anaemia34. Some of these studies have also reported that lower levels of vitamin B12 could result in macrocytic anaemia associated with reduced Hb levels35. Interestingly, majority of the AVNFH patients whose samples were analyzed in this study had significantly reduced levels of vitamin B12 compared to healthy controls. Although intriguing, additional validation studies are required to confirm‎ the association between vitamin B12 levels and reduced Hb in the context of AVNFH. In addition, random glucose levels were also significantly decreased in AVNFH patients compared to healthy controls. This is consistent with data from an independent study that describe significantly reduced levels of glucose in the synovial fluid of pigs wherein osteonecrosis was cryo-surgically induced36.

Our findings also suggest significantly higher levels of serum creatinine in AVNFH patients which could potentially be a result of muscle atrophy, a condition reported to be associated with AVNFH in long-term follow up studies in dogs37. In addition, an independent study has also described significant association between total plasma homocysteine levels and elevated serum creatinine in middle aged and elderly subjects38. Furthermore, in our study, plasma sodium levels were also higher in AVNFH patients compared to controls. Sodium levels in the range of 145–150 mM have been reported to induce secretion of von Willebrand Factor by endothelial cells leading to hypercoagulability and thrombosis39. In this context, it is important to note that late stage AVNFH is also categorized as a coagulation disease40.

The key finding of this study is the association of elevated levels of homocysteine with AVNFH. In parallel, levels of vitamins B12 and B6 as well as betaine are reduced. Importantly, this suggests that AVNFH could potentially be associated with altered methionine cycle, in turn resulting in elevated levels of homocysteine. Homocysteine is known to be an inflammatory agent. Multiple studies have demonstrated increased osteoblastic and osteoclastic activity in macrophages and bone cells treated with homocysteine. In line with this, our study further demonstrates existence of extensive bone remodelling in AVNFH. Additional in vitro and in vivo studies are necessary to unequivocally establish a causal relationship between homocysteine accumulation and bone remodelling in AVNFH. However, if validated, this would introduce the potential of testing inhibitors of methyltransferase or conventional DNMT1 inhibitors for the clinical management of AVNFH. In addition, ratio of homocysteine to betaine or homocysteine to vitamins B12 and B6, in serum could be eval‎uated for their potential to serve as non-invasive markers for early detection of AVNFH. In turn, development of such markers for early detection of AVNFH could provide an extended window for treatment thus reducing the number of debilitating hip replacement surgeries.

Betaine is a key substrate for the enzyme betaine homocysteine methyl transferase (BHMT) that converts betaine to methionine. Prior studies41 show that high levels of homocysteine in patients and mouse models with hyper-homocystinemia (diet induced or due to cystathione beta synthase deficiency) are associated with reduced levels of betaine. Consistent with this, an independent study demonstrated that betaine supplementation post methionine administration reduced total homocysteine concentration42. Taken together, these literature-associated data support our findings describing a reciprocal relationship between the levels of betaine and homocysteine in AVNFH. It will be important to determine whether lowering of betaine levels serves as a metabolic adaptation to maintain higher levels of homocysteine in AVNFH. Preliminary insight to suggest that this might be the case comes from our findings on reduced levels of vitamins B6 and B12, both of which serve as key co-factors in recycling of homocysteine to methionine. However, our data also demonstrates higher levels of methionine in AVNFH which is counterintuitive in the context of the above argument.

To understand this paradox, it is important to note that some of the earlier studies demonstrated normal to higher levels of methionine in majority of individuals with vitamin B12 or folate deficiency43,44,45. For example, serum levels of methionine were found to be normal or higher than normal in 51/60 patients with B12 deficiency and in 55/60 individuals with folic acid deficiency43. In an independent study44, methionine levels were predominantly higher than normal levels in cobalamin-deficient patients when compared to control individuals. Along the same lines, in an independent study45 levels of methionine in animals with vitamin B12 deficiency was similar to those observed in control animals, despite being fed methionine rich chow. Thus overall, these published literature shows that under conditions of vitamin B12 deficiency, methionine levels tend to be maintained within normal levels suggesting alternative mechanisms for methionine repletion such as increased dietary uptake of the metabolite that needs to be verified using additional studies.

All of the above clinical findings highlight the importance of studying altered metabolism in AVNFH. Elevated serum levels of homocysteine are known to be strongly associated with AVNFH27, while factors such as alcohol intake, smoking etc., that are known to be associated with onset of the disease, are known to alter circulating levels of vitamin B12 an important co-factor in the Methionine cycle. Furthermore, multiple studies have described mutations in MTHFR to be associated with AVNFH22, 46. Importantly, our plasma data suggests elevated levels of metabolites in the Methionine cycle leading to the formation of homocysteine while levels of compounds associated with recycling homocysteine back to methionine were significantly reduced. This is consistent with strong and significant negative correlation between levels of homocysteine and Vitamin B6/B12 in plasma samples. Together these strengthen findings describing elevated levels of homocysteine in plasma of AVNFH patients.

Prior studies have shown that higher levels of homocysteine could promote osteoclast formation47 resulting in increased bone loss as well as leading to thrombosis causing atherosclerosis and occlusion of arteries and veins48. According to the vascular hypothesis (or regional endothelial bed dysfunction), local microvascular thrombosis could decrease blood flow in the femoral head resulting in onset of AVNFH49, 50. Importantly, key metabolic alterations observed in AVNFH samples suggest occurrence of extensive bone remodelling in these samples. Thus for example, elevated levels of polyamines have been shown to be associated with increased osteoblast formation51, while elevated levels of SAM and SAH have been shown to induce osteoclast formation by promoting methylation-induced silencing of inhibitors of osteoclastogenesis52. Adenosine was also shown to be important for osteoclastogenesis53. Furthermore, reduced levels of Vitamin B12 have also been associated with osteoclast formation54.

Overall, our metabolic analysis highlights important vignettes that promote constant remodelling of bones, a characteristic feature of AVNFH.

AVNFH-associated bone remodelling is further substantiated by our biophysical studies. Micro-Raman spectroscopy is a widely used tool to study the chemical composition of the bone55. A healthy bone is composed of hydroxyapatite, carbonate substituted apatite and collagen56. Micro-Raman spectroscopy-based line scan analysis of AVNFH bones showed decreased Phosphate to amide I and carbonate to amide I ratio, suggesting reduced crystallinity, a measure of bone strength57. In addition, decrease in mineral matrix ratio in AVNFH bones that contain extensive breakdown of trabecular architecture, reveals extensive resorption of the necrotic bone. Furthermore, SEM images indicate a time lag between resorption and formation of new bone in AVNFH samples.

Interestingly, a more in depth Raman mapping analysis revealed an elevation in Carbonate to phosphate ratio in AVNFH areas when compared to controls. This is in line with the results of an earlier study where in necrotic bone of AVNFH was marked by increase in carbonate to phosphate ratio suggesting a trend towards increased resorption58. This is further supported by CT scan analysis of samples that were used for Raman mapping that revealed that the radio density (represented as Hounsfield number) associated with AVNFH was significantly lower than the values for the controls. Importantly, as reported by others, the mean number of HU within each region is a surrogate for bone mineral density59 and higher Hounsfield values are associated with tissues having higher density60. Taken together, these findings indicate extensive structural remodelling in AVNFH bone.

Remodelling in AVNFH bone was further verified using SEM. A hall mark of AVNFH bone when examined using SEM, is the appearance of dead bones and micro-cracks. Micro-cracks are microscopic cracks with length in the range of 30–80 microns61. The presence of trabecular micro-cracks in necrotic bone suggest healing defects and has been suggested to result in secondary vascular impairment in the capillaries either by causing compression of non-elastic fat cells or rupture of small intra-trabecular vessels62. Defect in bone structure was further visualized using histopathology that revealed absence of osteocytes within the necrotic zones. These could represent areas of dead bone as reported by others63. Importantly, necrotic areas containing dead bone appeared dark in SEM images and represented a unique feature seen only in AVNFH samples.

Despite these extensive changes in bone architecture, plasma levels of bone resorption markers like osteocalcin and parathyroid hormone (PTH) did not change in AVNFH patients compared to controls. Our results corroborate with that of earlier reports where in osteocalcin and a set of bone formation and resorption markers did not vary significantly between Osteonecrosis of knee and control patients64. Intriguingly however, osteocalcin expression‎ associated with osteoblasts in the sections of trabecular bones were significantly altered in AVNFH compared to controls. Interestingly, osteoblasts lining the bone in areas of necrosis or fracture repair (where extensive bone remodelling is known to occur) strongly stained for osteocalcin. These results are in line with previous reports that show increased osteocalcin deposition in AVNFH areas65. In contrast, osteoblasts in healthy bone or in regions distal from sites of necrosis or bone repair showed weak osteocalcin expression‎. Furthermore, CTX levels, an independent bone resorption marker showed a higher likelihood of being elevated in AVN compared to controls in a small pilot study that needs to be verified in larger patient and control cohorts. The relatively lower percentage of CTX positive AVN in our pilot study is consistent with a case report that describes absence of elevated CTX values in AVNFH that developed secondary to fibrous dysplasia66. Importantly, these findings suggest that AVNFH is more likely a localized disease with active remodelling of the bone occurring in specific areas associated with necrosis. Our immunohistochemistry data also showed significant non-specific intense staining in the marrow potentially due to high levels of peroxidase activity in these tissues. Yet another explanation for unchanged levels of bone resorption markers in the small group of AVNFH plasma that we analysed could be that these patients presented with advanced stage disease (stage 3 and 4).

Conclusions

Elevated levels of homocysteine and metabolites belonging to the homocysteine and polyamine pathway in the plasma are associated with Avascular Necrosis of the Femoral Head. The increase in homocysteine is correlated with a decrease in vitamin B12, B6 and betaine. Micro-Raman spectroscopy study shows that the bone from AVNFH patients displays decreased phosphate to amide and carbonate to amide ratio indicative of reduced mineralization. Raman mapping interestingly revealed a significantly higher carbonate to phosphate ratio, indicative of bone resorption, as well as decreased mineral to matrix ratio indicative of decreased mineralization in AVNFH bones consistent with the CT scan data demonstrating reduced bone mineral density. Consistent with all of this, SEM and histopathology reveal the presence of disrupted trabeculae and micro-cracks, co-existing with regions of dead bone as well as loss of osteocytes with associated necrosis of the marrow. Taken together, these data suggest extensive remodelling in AVNFH bones which is supported by increased expression‎ of osteocalcin in osteoblasts associated with necrotic sites.

Methods

Clinical Samples

Retrospective and prospective data on clinical parameters from AVNFH and controls were abstracted from the SSSIHMS database by a honest broker per approval of the SSSIHMS institutional bioethics commission (Approval number: SSSIHL/IEC/PSN/BS/2012/05) and provided for the study in a de-identified manner by a honest broker. Informed consent was obtained from all subjects. All samples used in this study were collected in a de-identified manner and the methods were carried out in “accordance” with the approved guidelines and regulations. Sections of trabecular bone or plasma from de-identified AVNFH patients were obtained from Sri Sathya Sai Institute of Higher Medical Sciences with approval from the institutional bioethics commission and patient consent. AVNFH trabecular bone sections (n = 13 total) were obtained post-hip arthroplasty. AVNFH plasma (n = 30 total) was obtained from patients visiting SSSIHMS for AVNFH treatment. De-identified control bone (n = 7 total) and plasma (n = 31 total) was obtained from fracture cases with no history of AVNFH. Paraffin embedded tissue sections for histopathology and immunohistochemistry from AVNFH and control samples were obtained in a de-identified manner from SSSIHMS and Baylor College of Medicine.

Analysis of Clinical Chemistry Data

Clinical data from 69 AVNFH patients (refer Table 1 for summary of clinical parameters) were used for the analysis. This included retrospective data from 55 patients and prospectively collected data from 14 patients currently consulting at the hospital. 1/69 patient was an outlier for all the clinical parameters and hence was excluded from all the downstream analysis. Clinical data from a random set of age and gender matched control individuals (n = 71) was also obtained in a de-identified manner.

Table 1 Information regarding the reference ranges of 9 clinical parameters in the plasma of AVNFH and Control patients.

Full size table

Out of a total of 23 clinical measurements that were obtained for each subject, only 9 parameters had values in at least 70% of the total subjects. For these parameters, missing values in patients or controls were computed using the median value for the entries in the corresponding experimental group (i.e., AVNFH or control). The levels for each of the parameter were then visualized relative to the corresponding clinical reference range. To determine the significance in the altered levels of parameters in AVNFH relative to control, a student t-test was applied. Box plots were used to visualize significantly altered parameters in AVNFH and control groups after removing one of the AVNFH outlier samples as described above. Following this, multivariate analysis was performed using PLS-DA on all the variables. Logistic regression model was built using all the variables including age and gender. This analysis used the Generalized Linear Model (glm) function in the Stats package in R. The predictive power for these three parameters was verified using a 10-fold cross validation repeated 1000 times.

Metabolite Analysis of Plasma samples using Mass Spectrometry

Plasma was extracted from blood samples collected in EDTA coated tubes following standard protocols. For extraction of metabolome, plasma samples were homogenized in 1:4 ice cold water: methanol mixture containing equimolar mixture of 2 standard compounds, Zeatine, [15N]2-Tryptophan. Extracts were de-proteinated by passing through a 3 KDa filter, filtrate was dried, resuspended in injection solvent (water: acetonitrile, 95:5, containing 0.1% formic acid) and analyzed by liquid chromatography-coupled to mass spectrometry (LC-MS/MS). The LC-MS/MS analysis was carried out on a Micromass Quattro Micro™, Waters Inc., Manchester, UK and Agilent 6420 triple quadrupole, Agilent Technologies, Santa Clara, CA coupled to Waters and Agilent HPLC system, respectively. Data acquisition was performed using MassLynx software (Waters) and Mass Hunter workstation software (Agilent). A detailed information regarding the operational parameters of HPLC and LC-MS/MS analysis for the samples is given in the Supplementary methods section.

Micro-Raman spectroscopy

Micro-Raman measurements were carried out using a Raman microscope (T64000-HORIBA Jobin Yvon, Kyoto, Japan) at Raman research Institute, Bengaluru, India. A Helium-Neon (He-Ne laser excitation wavelength of 633 nm) was used to minimize background fluorescence from the biological specimen. The excitation laser power incident on the sample was fixed at about 2 mW. Each spectrum was recorded using 1800 grooves/mm grating with 80 seconds of exposure time over the region of interest. Multiple Raman spectra were obtained along the diameter of the bone slice at 2600–3000 micron intervals, by focusing the laser beam through a 50XLWD (long working distance) microscope objective. For AVNFH specimens, the spectral acquisition spanned the entire diameter of the bone encompassing adjacent non-diseased areas and AVNFH containing regions. For control specimen, an equivalent number of Raman spectra were obtained along the diameter of the bone slice. Raman spectra were recorded between the wavenumber ranges of 400–1800 cm−1 to enable examination of both the mineral and organic phases of the bone tissue. For each of the bone slices, a line scan typically comprising of 10–12 spots was carried out along the entire cross section of the bone. All the data were normalized with respect to the peak intensity of phenyl alanine at 1002 cm−1. The normalized data was used for downstream statistical analysis to compare and quantify the mineral-matrix components in the AVNFH bone versus the control bone like bone crystallinity, Carbonate to amide ratio and Phosphate to amide ratios.

Raman mapping

Raman mapping was carried out using Thermo Scientific DXR Raman microscope emitting 780 nm infrared wavelength at Sri Sathya Sai Institute of Higher Learning, Puttaparthi, India. Laser beam was focused on the sample through 10X objective. Each spectrum was recorded using 400 grooves/mm grating with 10 seconds of exposure time with an estimated resolution between 4.7 and 8.7 cm−1 over the region of interest. The excitation laser power incident on the sample was fixed at about 4 mW. The typical spot size impinging the sample was 3.1 µm. The mapping was carried out on two morphologically distinct areas of AVNFH bone sample representing the necrotic and the non-diseased adjacent areas as determined by the clinician, using rectangular grids of 300 × 300 µm2. A similar area was also chosen in the control bone samples for analysis. Raman spectra were recorded in an extended spectral range between 100 and 3300 cm−1. Raman maps showing the intensities of phosphate, amide and carbonate functional moieties along with ratios for Carbonate to phosphate and mineral to matrix ratio were computed. Median values with associated standard deviations were used to compare between the groups. Two-sided t-test was used to determine the significance of the results.

Scanning Electron Microscopy (SEM)

The bone specimens stored in PBS at −20 °C were thawed and subjected to a pre-treatment process to preserve the integrity of collagen and remove any cellular and surface organic material, following protocol published by67. Notably, for the SEM analysis, the bone samples were not sputter coated so as to avoid any artifacts. Processed specimens were examined using a Carl Zeiss Ultra plus Gemini Electron Microscope (Oberkochen, Germany) at the Raman Research Institute, Bengaluru, India.

Computer Assisted tomography analysis of Hounsfield units (HU)

CT scans were acquired on multiple trabecular bone cross sections of AVNFH patients (n = 7) and healthy controls (n = 4), using GE Discovery CT 750 HD GST FREEDom edition CT scanner at the Sri Sathya Sai Institute of Higher Medical Sciences Prasanthigram, India. Parameter used to acquire the CT image included: Tube voltage of the CT-scanner: 140 kV, current: 280 mA, slice thickness: 0.625 mm and interval between slices: 0.625 mm. CT acquisition was carried out in high resolution mode using the application software 13MW24.7 V40-PS HD64-G-GTL. CT images were saved in DICOM format using the Synapse software version 4.3.2 Fuji film medical system. U.S.A. Inc. Multiple elliptical Regions of Interest (ROI), each having an area of 50 mm2 and perimeter size of 25 mm were superimposed on the reconstructed images of each of the bone slice using the synapse platform. CT intensities were computed in Hounsfield units (HU) for each of the ROIs. Median value of CT intensities was computed across all the bone slices independently for each patient/control sample.

Bone Histopathology and immunostaining for osteocalcin

Necrotic and normal adjacent areas from the bone slices obtained from AVNFH patients were grossly excised and fixed independently along with the control bone slices in 37% formaldehyde. Following this, all the specimens were decalcified using 10% EDTA, dehydrated, processed conventionally and embedded in paraffin wax. The embedded bone specimens were cut into 5 µm sections using a Leica microtome (Buffalo Grove, IL at M.S Ramaiah Medical College, Bengaluru, India. The sections were stained using Hematoxylin and Eosin and reviewed by two board certified bone pathologists from M.S Ramaiah Medical College, Bengaluru and BCM. These sections along with additional ones from BCM (5 AVNFH and 2 controls) were used for immunostaining using osteocalcin antibody. Prior to osteocalcin staining, tissues were de-paraffinized in xylene and rehydrated in graded alcohols. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide. Slides were blocked by 3% goat serum at room temperature for 1 hour in humidity chambers and incubated first with anti-rabbit Osteocalcin (Bioss Antibiodies Inc. Woburn, MA, USA) for 2 h and then with HRP conjugated goat anti-rabbit secondary antibody (Jackson Immunoresearch Laboratories Inc, West Grove, PA) for 40 minutes. The antigen-antibody reaction was visualized after diaminobenzidine (Sigma-Aldrich, MO) and the slides were counterstained with hematoxylin (Sigma-Aldrich, MO) and mounted with Permount media. Positive controls were stained in parallel; negative controls were generated by omitting the primary antibody.

Parathyroid Hormone (PTH) and Osteocalcin measurement in sera were carried out using the kits PTH 11972103160 and Osteocalcin 12149133160 (Roche diagnostics), following manufacturer’s protocols at Thyrocare Mumbai and Metropolis Mumbai respectively.

CTX (Beta-CrossLaps) measurement in plasma was carried out using the kit 11972308160 (Roche diagnostics), following manufacturer’s protocols at Metropolis Mumbai.

Experiments on humans and the use of human tissue samples. We confirm‎ that all experiments were performed in accordance with relevant guidelines and regulations.

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