Journal of Orthopaedics and Traumatology

COMPREHENSIVE STUDY OF BONE MARROW INJECTION IN MANAGEMENT OF

DELAYED UNION OF LONG BONES
--Manuscript Draft--

Manuscript Number:
Full Title: COMPREHENSIVE STUDY OF BONE MARROW INJECTION IN MANAGEMENT OF
DELAYED UNION OF LONG BONES
Article Type: Original Article
Keywords: bone marrow injection; delayed union; long bones.
Corresponding Author: Praveen Madan
KMC Mangalore
Mangalore, Karnataka INDIA
Corresponding Author Secondary
Information:
Corresponding Author's Institution: KMC Mangalore
Corresponding Author's Secondary
Institution:
First Author: Deepak Pinto
First Author Secondary Information:
Order of Authors: Deepak Pinto
Vivek Nahar
GVSN Raja
Praveen Madan
Order of Authors Secondary Information:
Abstract: Abstract
Background: Delayed union is commonly seen in diaphyseal long bone fractures. Bone
grafting can be easily done percutaneously and is increasingly being used to treat
delayed union. In this study we assess the osteogenic activity of bone marrow, by
percutaneous route which can be used as a substitute for operative bone grafting.
Material and Methods: In this study there were a total of 45 cases of delayed union
treated with bone marrow injection. We treated 39 tibias, 4 femur and 2 humerus
delayed unions. Intramedullary nailing was found to be the most frequently used
modality. Average time elapsed before bone marrow injection from the time of initial
fixation in our study was for tibia was 14- 20 wks, for femur it was 16-24 wks and for
humerus it was 12-18 weeks. The volume of the bone marrow injected was 40ml
Under C arm guidance. Results: Out of 45 cases treated with bone marrow injection in
our study, satisfactory union was seen in 42 cases. Only 3 cases went for non-union
for which open bone grafting was done. Average time to union in our study for tibia
(mean-12.2 wks), femur and Humerus it was 10- 19 weeks (mean-15 wks) and 7 - 14
weeks (mean-11 wks.) respectively.
Conclusion: Percutaneous autologous Bone marrow injection was relatively simple,
cost effective with least recipient and donor site morbidity. Thus it can be good
alternative method to bone grafting in the treatment of delayed union of long bones.
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Authors
Deepak Pinto
Vivek Nahar
GVSN Raja
Praveen Singh Madan




Title

COMPREHENSIVE STUDY OF BONE MARROW INJECTION IN
MANAGEMENT OF DELAYED UNION OF LONG BONES

Authors Details

Deepak Pinto
Dept of Orthopaedics
KMC Mangalore
deepakpinto@hotmail.com

Vivek Nahar
Dept of Orthopaedics
KMC Mangalore
drvpnahar@gmail.com

GVSN Raja
Dept of Orthopaedics
KMC Mangalore

Corresponding Author
Praveen Singh Madan
Dept of Orthopaedics
KMC Mangalore
Madan.praveen@gmail.com
91-9739529136
Title Page
Click here to download Title Page: title.docx
1
INTRODUCTION

Bone grafting remai ns among t he most frequent of orthopae dic
procedures ( Lane and Sandhu 1987). Bone grafts are used to fill
caviti es or defects resul ting from cyst s, tumors, to promot e uni on or
fill defect s in del ayed uni on, non-union, fresh fractures or ost eot omies,
to bridge j oints and thereby provide art hrodesis (Burchardt 1987,
Crenshaw 1992). [1, 15, 58]

Since the pioneeri ng work of Macewen in 1881 of reconst ruction
of t he di aphysis of t he humerus i n a young child, many subst ances have
been consi dered for use as impl ant or transpl ant mat eri al s. Current
opini on is, fresh aut ogenous bone is the most effective graft materi al
avail abl e for most cl inical situati ons (Lane and Sandhu 1987). [58]

The t echni que of bone grafting i nvol ves harvesting graft mat erial
from donor sit e, usual l y pel vis and operative impl ant ati on at t he
requi red sit e. Bot h t he procedure of harvesting t he graft and
implant ation are associ at ed with compl i cations (Covent ry and Tapper
1972, Lane and Sandhu 1972, Gershuni and Pi nsker 1982, Lauri e et al
1984, Summers and Eisenst ei n 1989). [29, 41, 58, 59] Jeopardizing t he
unaffect ed part, prolongi ng durat ion of surgery, painful scar,
hematoma, infection, fracture, gait dist urbances have been report ed
among t he problems at t he donor sit e. In addition the need t o open t he
fracture sit e has added to t he risk of i nfection or devascularisati on of
*Blinded Manuscript
2
the fract ure where heali ng is already impai red. Though recentl y
devel oped ost eogeni c mat eri als can overcome donor sit e morbi dit y,
they requi re sophist icated bone banking and are not freel y avail abl e
(Burwell , Friedlander and Manki n 1985). [17, 18, 58]

The research duri ng t he last cent ury has lead to the
understandi ng of t he t hree basi c mechanisms underl yi ng bone
regeneration vi z. osteogenesis, osteoinduct ion, and
osteoconduction. [58] Ost eogenesis is carri ed out - by pre-existi ng
differenti at ed or det ermi ned bone formi ng cell s. Autol ogous bone and
bone marrow graft s are exampl es of transplants with osteogeni c
properti es. Ost eoinduction ent ails di fferenti ati on of uncommitt ed
connecti ve tissue cells int o bone forming cells in t he presence of an
inductive sti mulus. Ost eoconductive grafts t ransfer neither
osteogeni ccells . nor inducti ve stimuli but behave as a non-viabl e
scaffol ding for the i ngrowt h of the vessels and new bone by Creepi ng
substi tution".

The abilit y of the bone marrow to sti mulat e ost eogenesis is
known for more than a century since t he experiment al works of Gouj an,
1869 (Connoll y 1995). The great versatilit y of bone marrow as a
transplant has been shown t o depend on the presence of st em cells. In
the bone marrow, osteogeni c stromal or stem cells are closel y
associ at ed wi th hemopoi eti c stem cel ls. The success of bone marrow
transplant i n hemopoi etic di sorders has l ead to invest i gations t o
3
det ermine whet her t heori es and t echniques l earned from hemopoi eti c
bone marrow transplant s coul d be appl ied to probl ems of skel etal
repair. The ost eogenic propert y of bone marrow has been proved in
experiment al animal s beyond doubt. [26, 30]

The t echni que of bone marrow grafti ng harvested by
percut aneous aspirat ion from ili ac crest (and the other sit es whi ch are
rich i n red marrow) and t ranspl anted t o the requi red si te through a
needl e offers to overcome most of the compli cations of the operative
bone graft ing menti oned before. The onl y di ffi cult y is i n retaining
the, bone - marrow at the required sit e. However t he delayed unions,
non-uni ons (even in infected cases when the sinuses don' t communi cate
with fract ure sit e) and cystic l esions, (t he cont ents of whi ch can be
evacuated through a needl e) provide t he ideal si tuations for bone
marrow grafting.

This study is one such at tempt t owards the assessment of
osteogeni c activi t y of bone marrow i n human beings whi ch can be used
as a substitut e for operati ve bone grafti ng.






4
AIMS AND OBJECTIVES

1. To Find out whet her Del ayed Uni on of Long Bones can be
successfull y t reat ed with Aut ologous Bone Marrow Injecti on
2. To st udy appropri ate time when a percutaneous bone marrow
injecti on should be given in management of del ayed union.
3. To assess the feasibi lit y of bone marrow injecti on as a substit ute for
bone grafting, clini cal l y and roentegenographi call y.
4. To assess average ti me t o union following bone marrow i nject ion
5. To assess the factors that i nfl uence the out come of bone marrow
injecti on
6. To find out suit able methodol ogy for bone marrow grafting to make
the procedure qui ck and effecti ve











5
MATERIALS AND METHODS

All cases of del ayed union t hat have been treat ed in the
department of Ort hopaedi cs, KMC, Mangalore duri ng June 2010 t o Oct .
2012 wil l be i ncluded in the st udy.
Ethi cal clearance was obt ai ned before the study was begun.
Type of study: Prospective.
Sampl e Size: 33
Durati on of st udy: June 2010 to Oct 2012

INCLUSION CRITERIA
Pati ents were included in t he study based on t he following cri teria:
1. Del ayed union: Union was considered to be del ayed when
healing has not advanced at t he average rat e, for the location and
t ype of fracture.
2. Age group 18-55 years

EXCLUSION CRITERIA
1. Del ayed unions with defect more than 1cm were excl uded.
2. Infecti ve del ayed uni on.
3. Age <18yrs and> 55 yrs.
4. Pathologi cal Fract ures.

All the pati ents were treat ed as In-Pati ents. Thorough cli ni cal
evaluati on was done and observations were not ed down accordi ng t o
6
the proforma. Cli ni cal diagnosis was confi rmed by roentegenographs.
Other investi gations done were compl et e hemogram, urine for albumin,
and sugar and microscopy. The investi gations requi red for the fit ness
for anesthesia were carri ed out as necessary.

Formal writt en consent was obtai ned from the patient/ guardian
aft er explaining t he procedure and its expect ed out come and
complicati ons. Clini cal photographs were t aken prior to the procedure
and duri ng t he follow up at important -st ages.

PROCEDURE: (Figure 1. a and 1. b)
With the pati ent under anest hesi a, eit her spinal or general, the
iliac regi on and t he part to be graft ed were prepared independentl y.
Then wit h the hel p of image i ntensifi er the fract ure site, was
visualized. The 16G bone marrow aspi rat ion needles with st i l ett e were
placed at the fracture site in an area where t here was adequat e soft
tissue covering (e. g. post erior surface of t ibia).
Then the bone marrow was aspirat ed wit h the hel p of anot her set
of bone marrow aspi rat ion needl e from the anterior superi or il iac spine
or posterior superi or iliac spine or t he adjacent ili ac crest (depending
upon the position of the patient ) in 10ml ali quot s from the punctures
placed at least 1cm apart . The bone marrow thus aspi rat ed was
immediat el y i nject ed at the required sit e through the needl e whi ch was
already pl aced with the help of image intensi fier. Care was taken to
inject the bone marrow before it clot ted. The volume of t he bone
7
marrow inj ected was 40ml in lower limb fracture healing problems. The
average time t aken for the procedure was 15-20 minutes. The steril e
dressi ngs were appl ied to both donor and reci pi ent area aft er the
procedure.

Post operative management:
During the post operative period all the pati ent s were prescri bed
anal gesi c and anti biotics. The immobi l ization of t he fracture was
achi eved by Pl aster cast whenever necessary.
Follow up :
The pati ents were followed up clini call y and
roent egenographi call y at 4-6 weeks int erval.
Method of Evaluati on of results of treatment:
The result s were graded as excellent, sat isfactory and fail ure as
per Hanson and Eppright (1966) crit eri a. [46]
Excell ent - Cases showing roent egenographi c evidence of union,
unprot ected full wei ghtbeari ng, wi thout pai n (painl ess use of the limb
in case of upper limbs) and subsi dence of infe ction i f present.
Satisfact ory - Cases with roent egenographic evidence of union and full
wei ght bearing (Painless use of t he l imb in case of upperlimbs) but for
whom, because of qualit y or quant it y of bone at the sit e of uni on, brace
prot ection was stil l considered necessary, or those with exacerbati on of
infection.
Failure - incl uding failure of union or amput ation.

8
REVIEW OF LITERATURE

ANATOMY OF BONE AND BONE MARROW STROMAL SYSTEM
Bone is a speciali zed connecti ve t issue with a mi nerali zed
coll agenous frame work for skelet al support of the body. Bone arises
by intramembraneous or endochondral ossifi cation.

MACROSCOPIC STRUCTURE
It i s either spongy (cancell ous) or compact in st ructure. Spongy
bone consists of inter crossing and connecti ng bone t rabecul ae of
varying shapes and thi ckness, bet ween whi ch are spaces filled wit h
bone marrow. Compact bone is a cont inuous bone mass cont aining
interconnecti ng vascular channel s of mi croscopi c size. Bot h spongy
and compact bone exist in almost every bone, though in varyi ng
proportions. A t ypi cal large bone consists of diaphysis (which is
mainl y made up of compact bone), epiphysis and met aphysis (which are
mainl y made up of spongy bone). The small bones and flat bones
consist a shell of cortical bone covering t he spongy bone.

MICROSCOPIC STRUCTURE
The Dut ch scientist Anton Van Loeuwenhock was the first to
describe bone st ruct ure. The basi c st ruct ural unit of bone is Haversi an
System (Ost eon). It contai ns l amell ae concentri call y arranged around
Haversi an canal. Haversi an canal communicat es wit h the lacunae whi ch
9
contai n ost eocyt es t hrough canali cul ae and with t he medull ary canal
and surface of the bone through Vol kmann' s canals.

BONE TISSUE AND ITS CONSTITUENTS
1. Periosteum : the periost eum is the membrane coveri ng the outer
surface of the bone. It is composed of t wo l ayers. The fi brous
layer, an out er thin layer of dense, irregul arl y arranged
connecti ve tissue contai ning some fibroblasts and the ost eogenic
layer cont aini ng ost eogeni c cell s which are fl att ened and spindle
shaped cells with pl uripotenti al.
2. Cel ls of the bone :
a. Ost eoprogenitor cel ls (ost eogeni c cell s) are normall y
found apposed to t he bone surface in the deep l ayer of
resting periosteum, and t hey also comprise the endosteum
where they are likewise apposed to t he surfaces. They can
differenti at e i nto osteobl asts, chondroblasts, fi brobl asts
and perhaps ost eocl asts.
b. Ost eobl asts - synthesize, and secret e the organic
intercel lul ar subst ance of bone with whi ch they surround
themsel ves: They come to li e within the lacunae and
become ost eocyt es.
c. Ost eocyt es-probabl y represent inactive osteoblasts.
d. Ost eocl ast - The osteocl ast consist s of a multi nucleat ed
gi ant cell varyi ng in size and number of nucl ei. It
10
functi ons t o resorb both t he mineral s and int ercell ular
organi c subst ance.
3. Inter cellul ar substance: The int ercell ular substance consists
mainl y of matrix, inorgani c salt s and wat er. Matrix is the organi c
framework consisti ng chiefl y coll agen and small amount of
pol ypeptides and some gl ycoprot ein. Inorgani c s alts consist
mostl y of submicroseopic cryst als of hydroxyappatit e. Wat er
occupies the spaces within the bone, including the nut rient
canals of t he Haversian syst ems and the ult ramicroscopic
channel s within the molecul ar aggregat es of the coll agen fibri ls.

STROMAL SYSTEM OF BONE AND MARROW
The st romal syst em of bone has been defined as those connective
tissue elements within the marrow cavi t y t hat provi de structural and
functi onal support for hematopoi esis (Beresford 1989, Diduch et al
1993). Fi brobl asts, ret icular cel ls, adipocyt es, ost eobl asts and bone -
lining cell s are those el ements. [10]

It has long been recognised t hat i n t he postnatal organi sm,
osteogeni c precursors exist in cl ose associ ation with the soft , fi brous
tissue of the marrow st roma (Fi g 1. ) (Nade 1970, Buring. K 1975,
Hirano and Urist 1981, Tri ffitt 1987). [16, 50, 66, 91]
Marrow cell suspensions of rat, mouse, guinea pi g, rabbi t and
canineori gin have all been shown to be capabl e of forming an
est eogenic tissue whent ransplanted i n-vivo wit hin di ffusion chamber.
11
BONE HEALING AND ITS ABNORMALITIES
(Delayed union)

Few subjects have commanded a great er share of surgical
literature t han the growth and repair of bone and many hundreds of
papers have been published. Bick in 1948 report ed an estimate of 5000
papers i n est abl ished journals (Wilson 1992). [7]

Ever since Hunter demonst rat ed that bone is a dynami c tissue
invol ved in the equilibri um of bone depositi on and resorpt ion, the
mechanisms of bone repai r have been st udi ed. Hunter described the
cl assi c stages of nat ural bone repai r:
(1)Infl ammati on (2)Soft callus (3)Hard callus (4)Remodelling.

Inflammation st age usuall y l asts 1 to 3 days, and is clini call y
evi denced by pain, swel ling and heat . Inflammatory cells arrive at the
injured sit e accompanied by vascular i ngrowth and cell ul ar
prol iferat ion. Stage of soft callus formation is charact eri sed by
ingrowt h of capill ari es i n to t he fract ure callus and t he appearance of
chondroblasts and osteobl asts (Axhausen 1956). In the st age of hard
callus the fibrocartil agi nous tissue is replaced by fi bro-osseous tissue,
clini call y thi s usuall y occurs at 3-4 months. The final stage of
remodelli ng begi ns with clini cal and roent egenographi c union and
persist s until the bone is ret urned to nor mal. [108]

12
Currentl y it is accept ed t hat the fract ure healing process is
ani nterpl ay of cell ular el ement s and humoral mediat ors. Cell ul ar
component s are mai nl y cont ri but ed by bone marrow st romal syst em,
cambium l ayer of peri ost eum, met apl asi a of sorrounding soft ti ssues
and di apedesis of cells from the circul at ing bl ood. Humoral medi ators
are contribut ed by the bone matrix, the ost eogenic cell s and t he
infl ammatory cell s.

A multitude of l ocal and syst emi c fact ors infl uence bone heali ng,
imbal ance of which may lead to delayed and non-uni on.



DEFINITIONS : Del ayed union - union is considered to be del ayed
when healing has not advanced at the average rate for t he location and
t ype of fracture (Wilson 1992). Non-uni on can be described as a
fracture in whi ch the reparative processes have come to a standstil l
with well defined roent egenographi c and hi stol ogi c si gns (Wilson
1992). [7]

ETIOLOGY: The exact causes of delayed and non-uni on are unknown.
In a revi ew of 842 pati ents wi th non-union of l ong bones Boyd,
Li pinski and Wiley found that non-union was more common when the
fractures were
13
(1) Compound (2) Infected (3) Segment al withi mpairedblood suppl y
(4)comminut ed by severe trauma (5) Insecurel y fixed (6) Immobilized
for an insuffi ci ent ti me (7) Treat ed by an ill advised open reducti on (8)
Dist ract ed ei ther by traction or by pl at e and screws (Boyd, Lipinski
and Wiley 1961). [13]

Other causes are:
1. Loss of blood suppl y eg. anat omi cal suscepti bilit y of lower third
of t ibi a.
2. Damage to surroundi ng soft tissue
3. Extensi ve gap due to loss of bone substance at the time of
fracture.
4. Abnormal biomechanics.
5. Met abolic di sturbances.











14
STUDIES ON TREATMENT OF DELAYED
UNIONS BY BONE GRAFTING

In t he year 1947, Phemist er demonstrated that there i s no
necessit y t o break the fi brous union, in fact doing so may be
count erproducti ve as the st abilit y given by fi brous union is lost. He
showed that the fibrous union can be convert ed in t o bony union by
cancell ous bone graft ing around the fract ure non-uni on site. This
techni que has come to be known as Phemister t echnique" named aft er
the invent or. [73]

In 1955, Jones and Barnett have report ed the results of t reat ment
of non-union of tibia by post erol at eral cancellous bone grafti ng. Twent y
two ununited tibiae i n 20 adults were t reated by this method. Sixt y five
procedures had already been done on t he involved extremi ties before
post erol ateral bone graft ing. Ten of t hese nonunionswere infect ed at
the t ime of bone grafting. In 9 of the non-unions the drainage st opped
foll owi ng thi s procedure and i n 15 l egs the fractures uni ted. Some of
these cases underwent secondary procedures for ski n coverage, impl ant
removal, tendon l engtheni ng, arthrodesis etc. , They confront ed 19
complicati ons viz. , i nfection of graft , angul ation of t he leg, circul atory
impai rment , pain, paresthesi ae, skin sloughing (Jones and Barnett
1955, Jones 1965). [55, 56]

15
Between 1952 and 1965 Hanson and Eppri ght treated 26 cases of
infected non-unions of t ibi ae by post eri or bone grafting. [46] The series
consist ed of 24 mal es, 2 femal es bet ween the age 16 t o 63 years, most
of t hem had sust ai ned automobil e accident, 23 of them were open
fractures, 3 were cl osed. They were of the opinion that, even though
the fract ure sit e i s i nfected the posterior compartment of l eg remains
steril e and a bone graft can be pl aced without unduerisk of infect ion
(Marmor 1967). Among t hem 25 cases were followed up compl etel y
and 22 successful unions were obt ained. They graded the resul ts as
excellent - comprising roent egenographic evidence of union,
unprot ected full wei ght bearing wi thout pai n, and subsidence of
infection if present, sati sfactory - comprising pati ents with
roent egenographi c evidence of uni on and full wei ght beari ng but for
whom, because of qualit y or quant it y of bone at the sit e of uni on, brace
prot ection was stil l considered necessary, or those with exacerbati on of
infection, fai lure-including fail ure of uni on or amputat ion. The result s
according t o these categories were Excell ent - 15, Satisfactory - 7,
Failure-3, Unknown-1.

In t he year 1969, Sout er report ed a ret rospecti ve study of 102
cases of del ayed union and non-union of long bone fract ures. Sixt y
seven were i nvol ving tibi al fractures 20 femoral , 12 radi us and ul na, 3
humerus. Onl y 26 cases were female, age ranging from 15-77 years.
Nearl y 70 per cent of these cases were treat ed conservativel y and 30
per cent were treated by int ernal or external fixati on. Most of th em
16
were closed fractures sust ained due to either domestic, i ndust ri al,
traffi c or sport s accidents. The duration bet ween initi al i njury to that
of bone grafti ng ranged from ei ght and hal f weeks to seven and half
years, They consi dered definit e mobilit y at fracture sit e beyond 12
weeks as pot ent ial del ayed union and advised bone grafti ng without
del ay. They used t he same technique as that of Phemi ster whil e
graft ing the bone at fract ure sit e. Ei ght y four percent of the fract ures
were unit ed withi n 16 weeks, and onl y 4 took longer t han 20 weeks,
complet e fail ure occurred in onl y one case probabl y because of
infection. [84]

In t he year 1975, Meyer et al have reported about the t reat ment
of infect ed non-union of fractures of long bones in 64 cases whi ch have
been foll owed up from 5 to 21 years. They t reat ed t hese cases by
debridement of soft tissue and bone and open sucti on irri gation
drainage, foll owed by ri gi d stabilization and cancel lous bone grafti ng
when indicat ed. They obt ained union i n 60 cases. They emphasize the
importance of local treatment of the infection prior to st abi lizing the
fracture fragments and the use of cancell ous bone graft s when
necessary. They concluded that most import ant factor in obt ai ning bone
healing in infected non union was ri gid stabi lit y of the fracture wit h
compression at fracture site (Meyer, Weiland and Wi llenegger
1975). [64]


17

BIOLOGY OF BONE GRAFTS
HISTORY
In 1668, the Dutch surgeon Job van Meekeren described the first
bone graft procedure (deBoer 1989). The first successful allograft was
performed in 1880 by Macewan from Scot land, to reconstruct the
segmental defect after resect ion of the osteomyeliti c humeral shaft
with t he resect ed wedges from-ost eotomies for deformi ties of rickets
pati ent s. [2]

Fol lowing t he i ntroducti on of asepti c technique of surgery in the
beginni ng of thi s century the development of bone graft surgery was
escalat ed. It was onl y aft er F. H. Albee' s work on bone graft surger y
was published i n Uni ted St at es in. 1915 t hat the Bone grafting began to
be increasingl y performed.

There i s an ever increasi ng demand for bone grafts fol lowing
thedevel opment of li mb salvage surgery.

USES:
1. To fill the caviti es or defect s from cysts, tumors or other causes
(eg. disease, t rauma)
2. To promot e union in non-uni ons.
3. To bri dge major defects and restore cont i nuit y of a long bone.
4. To bri dge joints for art hrodesis.
18
5. To provide bone blocks to limit j oint mot ion.
TERMINOLOGY :
An autograft is transplantat ion of a tissue or organ from one site
to anot her i n t he same person.
An isograft is a graft exchanged between peopl e wit h geneti call y
identical characterist ics (e. g. t wins).
An allograft is t ransplant ation from one person to another of
same speci es, but wi th di ssimil ar geneti c charact eri sti cs.
A xenograft is from one person of one speci es to a person of
anotherspeci es (e. g. calf bone to human).
Depending upon t he nat ure of the bone being t ransplanted it can
be eit her corti cal or cancell ous bone graft.

Types of bone graft:
1. Singl e onlay corti cal grafts
2. Dual onl ay grafts
3. Inl ay Grafts
4. Peg grafts
5. Medull ary graft s
6. Ost eoperiosteal graft s
7. Multipl e Cancell ous chi p graft s
8. Hemicyl indri cal grafts
9. Whole bone transpl ant

19
The aforementioned t ypes of bone grafts are nearl y i n their order
of evolution. This evolution has come t hrough an increasing knowl edge
of t he bi ology of bone grafts. Foll owi ng the work of Albee, a t hinner
inlay graft , cut with power driven saws with gl ass stopper precision
was the basis of bone grafti ng operati ons for t he fi rst half of t his
century. Neverthel ess the i nlay grafts were necessaril y sl ender, their
width couldnot be much greater than hal f that of the bones i nto whi ch
they were insert ed and t he fracture of the graft was not uncommon.
When inert alloys became availabl e, t here was a resurgence of t he use
of st ronger and wider graft s whi ch coul d be screwed on to the bone
with a di ameter atl east as great as that of t he host bone. Much then
depended on the strength of t he grafts as a means of i nt ernal fixation of
fractures quit e apart from any vit al capacit y it mi ght have, and t his
lead to the t reatment of un-unit ed fract ures by massive onl ay grafting
with screw fixati on. It was not until after the second worl d war that the
advantages of cancel lous grafts with thei r fast rat e of revascularizati on
and t hei r great er ost eogenic power was full y appreci at ed. This lead to
the slow abandonment of massive corti cal graft and the introduction of
met alli c internal fi xation as an adjunct t o c ancell ous graft ing of
unst abl e fractures (Wilson 1992). [7] At the end of t his stage of
evolution of bone grafti ng procedure, t he opini on of consensus was,
fresh autogenous bone is t he most effect ive graft mat eri al avail abl e for
most cl ini cal situat ions. When used for repai r of massive osseous
defect s resulting from trauma, infecti on, congenit al mal formations or
20
mali gnancy, however, autogenous bone has a number of major
disadvant ages.
1. The avail able suppl y of t ranspl antabl e tissue i s often insuffi ci ent
for l arge defects, particul arl y in children.
2. There is si gnifi cant risk of postoperative morbidit y at the donor
site (pain, hemorrhage, wound probl em, cosmeti c disabil it y,
infection, nerve damage) and
3. The abilit y to fabri cate a functional shape from t ransplanted
tissue oft en is limit ed, resulting i n l ess than optimal fil ling of
the defect.
In t he li ght of these complicati ons, numerous invest i gators in
thei r search for sui table alt ernatives to autologous bone, have explored
such diverse subst ances as homol ogous bone (allografts), heterograft s
(xenografts), demineral ized bone, deprot einat ed bone, and syntheti call y
derived organi c and inorgani c consti tuents of bone. Allografts,
although currentl y empl oyed as the most common alt ernati ve to
aut ografts, encount er the same immunogenic complicati ons as other
tissue allografts. Thus they are cl ini call y charact erized by signi ficant
rat es of fracture, resorpt ion, and non-uni on secondary to i mmunol ogi c
rej ection. Because of even poorer host tol erance, xenografts are l argel y
ineffective.

Three basi c machanisms underli e bone regenerat ion:
osteogenesi s, ost eoi nduction and ost eoconduction. Osteogenesis i s
carri ed out by pre existing di fferenti at ed or determi ned bone forming
21
cells. Autol ogous bone and marrow graft s are exampl es of transplants
with ost eogenic properti es. Ost eoinduct ion ent ails di fferent iation of
uncommitt ed connective tissue cell s int o the bone forming cells in t he
presence of an inductive stimulus (eg. Bone morphogeneti c
prot ei n). Osteoconductive grafts transfer neither osteogeni c cells nor
inductive stimuli but behave as non-vi abl e scaffolding for the ingrowt h
of vessels and new bone by "creeping substit ution" ie. they facilit at e
mi grati on of bone forming component s (Lane and Sandhu 1987). [58]

BONE GRAFT ALTERNATIVES

A number of osteogenic, ost eoi nducti ve and osteoconduct ive
subst ances currentl y are being invest i gated for use i n bone repair. It. is
considerable. that a sel ected combi nation of ost eogenic cel ls,
osteoinductive factors, and ost eoconduct ive mat rices can be combined
and fabricat ed in to implantable mat eri al custom suit ed to parti cul ar
clini cal demands.

Bone Matrix : The t issue constituent s of bone mat rix are t he cells and
extracell ular matrix. The mat rix ret ai ns the prot ei n bound calcium but
not bone mi neral. The major bone mat ri x component is coll agen. The
bone mat rix is obt ained by processi ng allogeni c corti cal bone. The
rat ional e-behind the use of bone mat rix is based on Urist ' s proposi tion
of a prot ein i n t he mat rix that has ost eoinducti ve properti es. This has
been mainl y used i n experiment al ani mals, though few human tri als
22
also have found it successful (Tuli and Singh 1978, Lance 1985,
Bol ander and Bali an 1986, Reddi, Wei rit roub and Muthukumaran 1987,
Aspenberg and Andolf 1989, Schmal zr ied, Gerald and Fi nerman
1993). [4, 9, 12, 57, 76, 92]

Bone morphogeneti c protein (BMP): In 1982 Uri st and hi s colleagues
report ed a modifi ed extracti on scheme and described a low molecul ar
wei ght bone morphogeneti c prot ein (BMP) fraction. BMP di ffers from
bone mat rix in several ways. BMP is cell free and i s solubl e in body
flui ds and doesnot incit e any det ect abl e cell mediat ed immune
response. The further development in BMP is devel opment of human
bone morphogeneti c prot ein (hBMP). The BMP has been found to be
useful i n bri dging skel et al defects in animal s. A few human clini cal
tri als also have been reported (Schmalzried, Geral d and Finerman
1993). [4]

Hydroxyappati te (HA) and tri cakium phosphate (TCP): several
formul ations of cal cium phosphat e are bei ng investi gat ed for use as
bone substit utes. These ceramics can be either compl et el y synthetic or
can be derived from a nat ural source. When syntheti c, can be formed
into di fferent shapes and sizes wi th varying pore sizes, the paramet ers
whi ch det ermine osteoconducti on, ost eoi nduction and mechani cal
charact eri sti cs. These bi oinert mat eri al s mainl y work by
osteoconduction (Schmalzri ed, Geral d and Finer man 1993). [4]

23
Though the bone mat rix, bone morphogeneti c prot ein and
bioinert ceramic mat eri al s (HA & TCP) have passed the clinical tri al s,
the compl exit y of the process of product ion and consequentl y t he cost
fact or are inhibit ive in thei r wide cl ini cal appli cation. . Bot h of t hem
work eit her by osteoinducti on or ost eoconducti on, and l ack any cells
directl y involved in osteogenesi s.

Bone marrow: The two aspects of t he bone regeneration have been
ful fill ed by discovering osteoinductive and osteoconduct ive bone graft
alt ernat ives. It wi ll. . . be rewarding to fi nd out a source of os teogeni c
cells whi ch can be used in combi nati on with these to enhance the
osteogenesi s furt her. Bone marrow used i n this context in combination
with various mat erials like, bone morphogeneti c prot ein, bone mat rix,
TCA, HA, coll agen, have been found to give satisfact ory results
(Muschler et al 1989, Ufist 1989). [3]

STUDIES ON BONE MARROW INJECTION

The bone marrow grafting has been used successfull y in
hematol ogi c conditi ons viz. , apl asti c anemia, leukemi a. The pioneeri ng
work in thi s fi eld lead to the Nobel prize recentl y awarded to
E. Donnall Thomas of t he uni versit y of Washi ngt on (Vaughan 1981,
Connoll y 1995). [26, 93] Thi s has prompt ed t he work on eval uati on of
methods and t echni ques of preparing an inject abl e ost eogeni c graft . A
number of invest i gat ors have specul ated on the useful ness of marrow as
24
a source of substit ut e of bone grafting mat eri al (Chapman 1980, Heal ey
et al 1990, Sharma et al 1992). [21, 38, 48, 102]
Burwel l in 1964, recorded t he enhanced osteogenesi s in
composit e grafts of aut ogenous marrow with all ogeni c cancellous bone
over all ogeni c cancellous bone al one. [17, 18, 19]
In 1970, Phili p J Boyne corici eved t he. idea of using bone
marrow-al ong with parti cul at e cancel lous bone and found it t o be
superi or t o that of using a solid bone with bone marrow in
experiment al animal s. [14]

In 1973, Simmons et al by aut oradiographi c observation of the
composit e grafts of bone marrow and autograft cont aining radioacti ve
nuclei delabel led marrow cells and ost eocyt es confi rmed t he earli er
view of Burwell t hat labi le ost eogenic precursor cell s in autogenous
marrow cont ribut e to ost eogenesis. [83]

In 1978, Salama et al used composit e graft of autogenous marrow
and Keil bone (xenograft ) i n their cli ni cal t rial and found satisfactory
results (Sal ama and Weisman 1978, Graham 1982). [77]

In 1981, Jackson et al used bone marrow grafting successfull y i n
secondary closure of al veolar palat al defects in chi ldren. [53]

Bone marrow stromal cells include bot h pre-existing ost eogenic
precursor cells and mesenchymal cell s that respond to BMP by
25
differenti ati ng int o osteobl ast s for the repair of large bone defects
(Nade and Burwell 1977, Takagi and Urist 1982, Wittbj er et al 1983,
1984). John cummi ne et al i ndi cated the possi biit y of bonel ess
bonegrafti ng i n t he form of marrow (Cummine, Armstrong and Nade
1983). [66, 67]

Bone marrow was used for ost eogenesis along wit h cerami cs viz. ,
hydroxyappatit e and tri calci um phosphate (Nade et al 1983). It has
been found out t hat when bone marrow i s used along with bone graft
substi tut es viz. , DBM, BMP, ceramics, xenografts they act
synergisti call y. (Nade and Burwell 1977, Takagi and Urist 1982,
Wittbjeretal 1983, Green, Hint on and Tri ffit t 1986, Gol dberg and
Capl an 1989, Weint roub et al 1989, Grundel et al 1991, Tiedemanet al
1991). [66, 67, 44, 90]

Bone marrow was used successful l y to bridge the bone defect s in
experiment al animal s by percut aneous injecti on by Paley et al in
1986. [72]
James. T Tri ffitt reviewed the li teratur e about cell s and other
fact ors i nvolved in i nitiation and enhancement of bone formation. The
stromal system of bone marrow has been defined wit h further cl arit y t o
be cont aini ng ost eogeni c stem cells (Burwell 1985, Fri edenst ei n,
Chail akhyan and Gerasimov 1987, Tri ffit t 1987, Beresford
1989). [10, 17, 18, 91, 107]

26
In 1989, Connoll y et al have tri ed to develop an ost eogeni c bone
marrow preparat ion as a substi tut e for bone grafts. They have used it i n
percut aneous inj ecti on form, whereby t ri ed t o reduce the surgi cal insult
to the tissues surrounding the needed site of ost eogenesis. Various
methods of concentration, cl ini cal appli cation and evaluati on have been
studi ed. [26]

The first si gni fi cant cli ni cal use of bone marrow graft as a
substi tut e to operati ve graft came in 1991, when John F. Connoll y and
his coll eagues used autol ogous bone marrow inj ection to stimul ate
healing in 20 un-united ti bial fractures over a fi ve year period.
Ninet een of these fractures were ori ginall y' compound fract ures and 10
were infect ed at the time of bone marrow inj ection. [27] Conventional
treatment had fai led in many of these cases. They inject ed the bone
marrow percutaneousl y aft er aspi rating it from post erosuperior or
ant erosuperi or ili ac spine and al ong t he iliac crest in 10ml . aliq uots
(tot al about 100-150ml). In 3 cases they used it wit h DBM powder and
when open reducti on was done, clot ted marrow was appli ed to the
fracture sit e. Most of. the ti mes it was done as an outpati ent procedure
under general anest hesi a. In hal f of t hecases t he
fractureswereimmobi lisedwit hpl ast er cast , and another, hal f byi nt ernal
fixation with intramedullary nail . Nine fract ures treat ed by plast er cast
immobilization and all the 10 cases' t reated by int ramedul lary, nailing.
in conjunction wit h bone marrow graft ing unit ed. These resul ts are
27
comparabl e t o that of aut ogenouscancell ous bone grafting but wi thout
the disadvant ages associ at ed wit h t he latt er. [26]

In 1993, Garg, Gaur and Sharma of Jawaharalal Institut e of Post
Graduat e Medi cal Educati on and Research, Pondicherry st udi ed the
effect of percut aneous autogenous bone marrow grafting in 20 cases of
un-unit ed fractures. Fifteen of the 20 cases were tibial non-unions,
average time from injury t o bone marrow grafti ng was 10 (6-18)
months. The procedure was performed under general anest hesi a and
bone marrow was aspirat ed from post eri or superior ili ac spi ne i n 5ml
aliquots. Two inj ecti ons of 15-20 ml of bone marrow 4-6 weeks apart
were used. Post operativel y t he fract ures were immobilized wi th plast er
casts in 17 of the 20 pat ients and external fixator followed by pl ast er
cast was used inremaini ng 3 cases. Pati ents were kept non wei ght
bearing for 6 weeks post operativel y, foll owed by prot ect ed wei ght
bearing unti l union. Sevent een of the 20 non-unions heal ed i n 5 (3-7)
months. [38]

In 1995, Garg and Gaur report ed a case of congenital
tibialpseudoart hrosis treated successful l y by percut aneous autogenous
bone marrow graft ing. Previ ous treatment of excision of
pseudoarthrosi s, int ramedull ary fi bul ar graft and aut ogenouscancellous
graft had fail ed to achieve uni on in t hei r case. [39]

28
In 1996, Loki ec et al treat ed 10 cases of active simpl e bone cysts
in 10 children wit h percutaneous aut ologous -marrow grafting aft er
aspi rating t he fl uid cont ent of the cyst and breaking the i nner lining of
the cyst cavit y wi th the needl e used to aspi rat e the fluid. All the 10
cases consoli dat ed roentegenographicall y and showed remarkabl e
remodelli ng wit hin 4 months. No complicati ons were encount ered
(Loki ec et al 1996, Lubicky 1997, Schr euder and Veth 1997, Cohen
1997). [60, 61, 81]

In 1997, Thakur et al have report ed t he use of percut aneous bone
marrow injecti on in preventi on of non-union. Ni nt een pati ents wit h
del ayed and non-union of fract ures were t reated, of whi ch 9 were
invol ving tibi a and 8 involvi ng femur and the remai ning 2 had fractures
of humerus. Operati ve t reatment was undert aken for all t hese pati ent s.
All t he pati ents were showing poor heal ing at 16 weeks (10 -22 weeks)
post fixation . The bonemarrow inj ect i on was done under gene ral
anesthesia, marrow was aspi rat ed from post erol ateral iliac wing and
simult aneousl y inj ect ed at requi red sit e. Ei ght y to 120ml of bone
marrow was i nj ect ed. Post operativel y no additional immobilizati on
was necessary and were fol lowed radiologi cal l y at 1 mont hs interval.
Union was achi eved in all t he pati ents. [89]




29


RESULTS
I Age
Table I
Frequency Percent
25 and
bel ow
6 13. 3
26 35 16 35. 6
36 45 13 28. 9
46 55 10 22. 2
Tot al 45 100. 0



Table II Sex
Frequency Percent
F 13 28. 9
M 32 71. 1
Tot al 45 100. 0





30
Table III Bone Involved
Frequency Percent
TIBIA 39 86. 7
FEMUR 4 8. 9
HUMERUS 2 4. 4
Tot al 45 100. 0














Table IV Method of Fixati on

Frequency Percent
IM NAIL 34 75. 6
EXFIX 9 20. 0
DCP 2 4. 4
Tot al 45 100. 0
31

Table V Average ti me to union V/ s. Age:



Table VI Average ti me to union V/ s. Sex


0 2 2 2 6
.0% 33.3% 33.3% 33.3% 100.0%
.0% 13.3% 12.5% 20.0% 13.3%
3 9 3 1 16
18.8% 56.3% 18.8% 6.3% 100.0%
75.0% 60.0% 18.8% 10.0% 35.6%
1 3 7 2 13
7.7% 23.1% 53.8% 15.4% 100.0%
25.0% 20.0% 43.8% 20.0% 28.9%
0 1 4 5 10
.0% 10.0% 40.0% 50.0% 100.0%
.0% 6.7% 25.0% 50.0% 22.2%
4 15 16 10 45
8.9% 33.3% 35.6% 22.2% 100.0%
100.0% 100.0% 100.0% 100.0% 100.0%
25 and below
26 - 35
36 - 45
46 - 55
Age
Total
6 - 10 10 - 14 14 - 18 18 - 20
Average time to union (in w ks.)
Total
.043 sig Fisher's Exact Test
p value
1 3 6 3 13
7.7% 23.1% 46.2% 23.1% 100.0%
25.0% 20.0% 37.5% 30.0% 28.9%
3 12 10 7 32
9.4% 37.5% 31.3% 21.9% 100.0%
75.0% 80.0% 62.5% 70.0% 71.1%
4 15 16 10 45
8.9% 33.3% 35.6% 22.2% 100.0%
100.0% 100.0% 100.0% 100.0% 100.0%
F
M
Sex
Total
6 - 10 10 - 14 14 - 18 18 - 20
Average time to union (in w ks.)
Total
32



Table VII Average ti me to union V/ s. Bone Invol ved



Table VIII Average ti me to union V/s. Method of fixati on
.814 NS Fisher's Exact Test
p value
4 13 12 10 39
10.3% 33.3% 30.8% 25.6% 100.0%
100.0% 86.7% 75.0% 100.0% 86.7%
0 0 4 0 4
.0% .0% 100.0% .0% 100.0%
.0% .0% 25.0% .0% 8.9%
0 2 0 0 2
.0% 100.0% .0% .0% 100.0%
.0% 13.3% .0% .0% 4.4%
4 15 16 10 45
8.9% 33.3% 35.6% 22.2% 100.0%
100.0% 100.0% 100.0% 100.0% 100.0%
TIBIA
FEMUR
HUMERUS
Bone
Involved
Total
6 - 10 10 - 14 14 - 18 18 - 20
Average time to union (in w ks.)
Total
.040 sig Fisher's Exact Test
p value
33


Table IX Period elapsed before first injecti on and Average ti me to
union V/ s. Age of pati ent:


Age N
Mi ni mu
m
Maxi mu
m Mean
St d.
Devi at i o
n Medi an
ANOV
A F p val ue
Per i od
el apsed
befor e
1st
i nj ect i o
n ( i n
wks. )
25 and
bel ow
6 12 20 16. 67 3. 266 17. 00 . 087 . 967
26 -
35
16 12 20 16. 13 2. 579 16. 00 NS
36 -
45
13 12 20 16. 15 2. 996 18. 00
46 -
55
10 12 24 16. 60 3. 777 17. 00
Tot al 45 12 24 16. 31 2. 983 16. 00
Aver ag
e t i me
t o
25 and
bel ow
6 12 20 15. 33 3. 266 15. 00 3. 801 . 017
26 - 16 8 18 12. 25 3. 000 12. 00 si g
2 9 14 9 34
5.9% 26.5% 41.2% 26.5% 100.0%
50.0% 60.0% 87.5% 90.0% 75.6%
2 4 2 1 9
22.2% 44.4% 22.2% 11.1% 100.0%
50.0% 26.7% 12.5% 10.0% 20.0%
0 2 0 0 2
.0% 100.0% .0% .0% 100.0%
.0% 13.3% .0% .0% 4.4%
4 15 16 10 45
8.9% 33.3% 35.6% 22.2% 100.0%
100.0% 100.0% 100.0% 100.0% 100.0%
IM NAIL
EXFIX
DCP
Method of
Fixation
Total
6 - 10 10 - 14 14 - 18 18 - 20
Average time to union (in w ks.)
Total
.200 NS Fisher's Exact Test
p value
34
uni on
( i n
wks. )
35
36 -
45
13 6 20 13. 85 3. 508 14. 00
46 -
55
10 10 20 16. 40 3. 098 17. 00
Tot al 45 6 20 14. 04 3. 503 14. 00








Table X Peri od elapsed before first injecti on and Average ti me to union
V/s. Sex of pati ent:


Sex N
Mi ni mu
m
Maxi mu
m Mean
St d.
Devi at i o
n Medi an
t
val u
e
p
val u
e
Per i od el apsed
befor e 1st
i nj ect i on ( i n
wks. )
F
13 12 18 14. 92 2. 253 14. 00
2. 06
1
. 045
M 32 12 24 16. 88 3. 087 18. 00 si g
Tot a
l
45 12 24 16. 31 2. 983 16. 00
Aver age t i me
t o uni on ( i n
wks. )
F 13 6 20 14. 62 3. 776 14. 00 . 693 . 492
M 32 8 20 13. 81 3. 421 14. 00 NS
Tot a
l
45 6 20 14. 04 3. 503 14. 00



35


Table XI Period el apsed before first i njection and Average ti me to
union V/ s. bone involved:







Table XII Period el apsed before first i njection and Average ti me to
union V/ s. method of fixati on:






39 12 20 16.05 2.772 16.00
4 16 24 19.50 3.416 19.00
2 12 18 15.00 4.243 15.00
45 12 24 16.31 2.983 16.00
39 6 20 14.10 3.669 14.00
4 14 16 15.00 1.155 15.00
2 10 12 11.00 1.414 11.00
45 6 20 14.04 3.503 14.00
Bone Involved
TIBIA
FEMUR
HUMERUS
Total
TIBIA
FEMUR
HUMERUS
Total
Period elapsed bef ore 1st
injection (in wks.)
Average time to union (in
wks.)
N Minimum Maximum Mean Std. Deviation Median
34 12 20 15.94 2.849 16.00
9 14 24 18.00 3.000 18.00
2 12 18 15.00 4.243 15.00
45 12 24 16.31 2.983 16.00
34 8 20 14.71 3.186 14.00
9 6 20 12.22 4.177 12.00
2 10 12 11.00 1.414 11.00
45 6 20 14.04 3.503 14.00
Method of Fixation
IM NAIL
EXFIX
DCP
Total
IM NAIL
EXFIX
DCP
Total
Period
elapsed
bef ore 1st
injection (in
wks.)
Average time
to union (in
wks.)
N Minimum Maximum Mean Std. Deviation Median
36











In this study t here were a tot al of 45 cases of del ayed union treat ed with
bone marrow i nj ecti on, out of which 32 were mal es and 13 were femal es.
Youngest pati ent was of 18 years and ol dest pati ent was of 54 years. Mean
age was 36. 48 years. Maximum number of pati ent s were i n 31-40 yrs age
group (n= 19). We treat ed 39 ti bias, 4 femur and 2 humerus del ayed
unions. Amongst various methods of fracture fixation used, Intramedul lary
naili ng was t he most frequentl y used modalit y (n=34). Ot her methods used
were Dynami c compression pl at e fixation (n=2) and external fixator (n=9).
Average time elapsed before bone marrow injecti on from the time o f ini tial
fixation in our st udy was for tibi a was 14- 20 wks, for femur it was 16-24
wks and for humerus it was 12-18 weeks. Once bone marrow i njection was
gi ven, pati ent s were followed up for si gns of union on 6
t h
, 10
t h
, 14
t h
week
and 6 weekl y thereaft er. Out of 45 cases treated wit h bone marrow
injecti on in our study, satisfactory uni on was seen i n 42 cases. Onl y 3
37
cases went for non-union for which open bone grafting was done. Average
time to union i n our st udy for tibi a was 8 - 16 weeks (mean-12. 2 wks)
foll owi ng bone marrow injecti on. And for femur and humerus delayed
unions it was 10- 19 weeks (mean-15 wks. ) and 7 14 weeks (mean-11
wks. ) respectivel y.
Age of the pati ent had a st atist icall y si gnifi cant relat ionship with ti me t o
union.




DISCUSSION

The bone marrow Mesenchymal St em Cells (MSC) are responsibl e for
invoking t he process of healing. These cells bel ong t o the renewi ng
popul ati on in an adult marrow and repl ace cells lost by se nescence,
mi grati on or inj ury. They are multi potent and have the capacit y to
regenerat e into t hemselves or other cells throughout life.
In the biology of healing, repai r begi ns with i nfl ammati on. Foll owi ng
infl ammation, by the process of chemot axis the cell s are at tract ed to the
site of injury and t he repai r proceeds in a met hodical manner. Thus, a
biological cascade of heali ng begins i n which platel ets play a major
rol e. The fi rst st age i s the st age of haematoma. Haemat oma perhaps acts as
a vehi cle to deli ver the cell s to the si te of i njury and begin t he process of
healing. The pl at el et s deli vered not onl y cordon off the area by forming a
38
clot and defini ng it but also help i n creating a mi croenvi ronment for the
process of heali ng t o proceed unimpeded. They recruit mesenchymal cel ls
whi ch are pl uri pot ent (the precursors of monocyt es, macrophages,
fibroblasts, osteobl asts et c. al so produce coll agen I and II). The
macrophages cl ear t he debri s and prepare the sit e for the healing process.
The release of VEGF, TGF, PDGF et c by the cell s that appear at the site of
infl ammation promot e angiogenesis, coll agen synthesis and formation of a
scaffol d. This rol e of platel ets in the i nitial st age of heali ng formed the
basi s of devel oping Plat elet rich Pl asma gel (PrP- gel) and i ts appli cation
in fract ure heal i ng. But lit tle i mportance was given to the
microenvi ronment or the medi um in whi ch this PrP- gel should function.
The fact that the first step of heali ng is formation of a fract ure haematoma
is almost forgott en in these procedures. So also is t he fact that pl at el ets
begin t heir function in a haemat oma first by organizing it. Open surgi cal
procedures for fract ure fixation are a wi tness to this phenomenon wherein
surgeons have observed t he various st ages of haemat oma formation which
is proporti onal t o duration of the fracture. Thus the heali ng cascade after
gi ving PrP-gel we presume begins from where it stopped perhaps i n a
microenvi ronment which is not very conduci ve.
Unlike the above, when whol e bone marrow is inj ected it initiates t he
cascade of heali ng from the beginning by forming a haematoma and a clot .
The clot forms a fibrin mesh withi n whi ch t he healing process begi ns by
infl ux of inflammat ory cell s and ingrowt h of fibrobl ast s and new capill ary
vessel s.
It i s a well -established fact that loss of haemat oma is one of t he causes for
39
non and del ayed uni on in open fractures. Hence the pri nci pl e of converting
an open fract ure t o closed fract ure as earl y as possi bl e deri ves its
importance and so i s the importance of haemat oma formation.
Multipl e punct ures done pri or to i njection i n our study produce
infl ammation and haematoma and creat e a favourabl e mi croenvironment for
the aspi rate t o be inj ected. The marrow aspirat ed also remai ns in a simil ar
microenvi ronment both before duri ng and aft er i njecti on. This procedure
closel y mimi cs t he biologi cal process of healing providi ng cell s and
growth fact ors in adequate amount for initi ation and conti nuati on of the
healing process.
The aspirat ed specimen of marrow whi ch is coll ect ed and i nject ed
immediat el y is presumed to cont ain adequate number of cel ls and fact ors
necessary t o initi at e heal ing for that individual case. Haemopoetic syst em
is a refl ecti on of marrow st roma. So i f the routi ne blood i nvesti gati ons
such as Hb%, TLC, DLC and pl at elet count are normal it refl ects t he
healthy st at e of the marrow.
Further, animal studi es by Ill izarov showed that bleeding ani mals i ncreased
the ost eogenic response to fill 5-mm fibular defect s when compared wit h
control animals that were unbl ed. Whi ch means that i n open fract ures or i f
fresh bl eeding is induced, t he bone marrow remains in a state of
preparedness ready t o promot e ost eogenesis. Thus eval uation of the st at us
of the marrow perhaps is not necessary prior to aspirati on because al l cases
studi ed were open fractures ini tiall y.
Since the marrow aspiration was in 3 ali quots of 10ml each over aspi rat ion
and dil ution was prevented. Among t he t hree aspi rates, t he fi rst aspirat e is
40
presumed to have high concent rat ion of cells and factors and t he last
aspi rat e proportionat el y less. There was no way to anal yze t hese aspirat es
because onl y adequate amounts were coll ected and inj ected. If the mat eri al
was taken for anal ysis it would result i n some loss of the aspi rat e whi ch
mi ght adversel y affect t he out come which is et hicall y unjusti fied.
No anti coagul ants were used in the procedure in order to promote adhesi on
whi ch i s the fi rst step of any heali ng process. Perhaps, i t is correct to
postulat e at this junct ure t hat in the concent ration t echni que where i n
anti coagul ants are used, this adhesi ve process is compromi sed. Furt her,
animal studi es and clini cal cases of non-uni ons treated with marrow
injecti on have shown that there is t endency for the marrow inj ect ed to
diffuse widel y away from t he area of i njection i f bindi ng does n ot t ake
place. This is t rue for fract ure of long bones li ke ti bi a where the area to be
inject ed is l arge t han i n a small bone li ke scaphoi d. Additi on of
anti coagul ants perhaps will facilit at e this diffusi on. It also prevents
formation of clot and demarcati on of the area. Hence, to initi ate t he
healing process more number of cell s i n a concent rated fashion may be
needed as some of t he cells may diffuse away. There is no evi dence til l
dat e i n humans t o prove the fact that in an arti fi cial mi croenvironment how
many of the cells in the t echni call y prepared concent rat e survive and
functi on effectivel y i n vivo foll owi ng inj ection.
In contrast , the nat ural process may not need cells i n such l arge numbers
because they are inject ed along wit h the medium in thei r nat ural
microenvi ronment and may not diffuse away.
The rati onal e for topi cal use of pl at el et -enri ched preparations is to
41
stimul at e the natural healing cascade and tissue regeneration by a supra -
physi ological rel ease of pl at elet -derived factors directl y at t he sit e of
pat hology. The amount of growt h factor availabl e for ti ssue healing
depends on the amount of growth fact ors actuall y stored in plat el ets and
the kineti cs of the adsorption of t he pl at el et gel .
Studi es show t hat t he growth factor content of the gel varies with the
number of plat el ets it cont ains and the method employed for processi ng.
Because of t his vari able nature, results become unpredict abl e. The
procedure does not give any import ance t o the clot ting cascade.
Considering the successful result s obtai ned in our study, we are forced to
rai se a question. Do we have to isol ate the cells, factors and devise more
sophi sti cat ed met hods to achieve uni on biologi call y? Inst ead is it not
possi ble to dupli cat e the natural biologi cal process from the begi nni ng?
Nishi moto et al in their study observed t hat the bone marrow cells t oo have
an import ant rol e to play in the healing process al ong with plat elets and
recommended a preparati on of PrP-gel and bone marrow cells which act as
working cells for si multaneous use.
Aspi ration of marrow in l arge amounts (50-150 ml) and inj ecting in
aliquots of 3-5ml in ununi ted fractures was recommended by Connoll y J et
al bet ween 1985 and 1990. [26] Lat er t hey recommended i njection of the
sedi ment contai ning cells obt ained aft er cent ri fugi ng t he marrow.
Our st udy of low vol ume bone marrow injection in tot o showi ng successful
results perhaps st ands as a t est imony t o the fact that heali ng is brought
about by groups of cell s; the parent cell being the bone marrow
Mesenchymal Stromal Cell (MSC). They bri ng about necessary changes
42
syst ematicall y at the sit e of pat hology from ti me to time ri ght from
scavenging and cl earing the dead and damaged ti ssue t o sealing the area
and fi nal l y healing and remodeling. Hence to achi eve a sound he ali ng
there is a need to provide everyt hing that i s necessary at the site of
pat hology. The si mplest way t o accompli sh thi s is t o transport it from t he
site where it is stored to the sit e where it is needed. The bone marrow when
aspi rat ed and i nj ect ed in t ot al (before cl otting) accomplishes this need. It
perhaps ini tiates the basi c process of adhesion, imbibati on, inoscul ation
and finall y upt ake in an orderl y fashi on similar to any other graft upt ake
by the process of chemotaxis. Maint enance of nat ural micr oenvironment
and a biol ogi cal medi um is absolut el y essential for t his t o take pl ace.
Recentl y animal st udies in rabbits using magneti c fi elds (magnet ot axis)
have been successful in transporting t he Ferumoxidel abell edMesenchymal
Stromal Cells t o the graft and enhance healing. The princi pl e empl oyed is
to facilit at e adhesi on. Adhesi on is an i mportant phenomenon. Once thi s
occurs other st eps should follow automat i call y.
We lay st ress upon t he t echnique whi ch has gi ven us successful result s. We
recommend that t he case sel ection crit eri ae and the steps mentioned should
be st ri ctl y be followed by t he t eam for successful results. We hope that this
simpl e procedure wi ll be t ri ed universal l y and adopted soon for treating
those ununi ted fract ures without defect and persist ent infect ion and more
morbid procedure such as bone grafti ng will remain as second line
procedure.


43







CONCLUSION

1. To concl ude t hat our study had est ablished that bone marrow
injecti on has hi gh osteogenic potenti al and can be grafted
successfull y i n cases of del ayed union of long bones.

2. In our st udy factors affecting uni on following bone marrow
injecti on were age of the pati ent , method of fixati on and time of
bone marrow inj ection and length of bone gap.

3. Appropri at e ti me for bone marrow inj ecti on in del ayed uni on of
tibia is found to be 3 mont hs and in del ayed uni on of femur was
4 months following fract ure fixati on

4. The average t ime of union following Bone Marrow inj ection i n
case of fracture of ti bia was 14. 1 weeks , in fracture femur is 3
months

5. Bone gap of more than 1cm were excluded from our study, as a
result effecti veness of bone marrow inj ection more t han 1cm gap
could not be assessed.

6. This minimall y invasive procedure is a bi ologi cal method of
bone grafting as it does not disturb t he vascul arit y of bone and is
rel ati vel y simpl e t echnique where del ayed uni on responded by
forming cal lus in t he area where bone marrow was i nj ected

44

7. In our st udy Bone marrow i nj ecti on was t aken from ili ac crest
and int roduced i nto t he fract ure site under C arm guidance
, met hod was rel ativel y simple , cost effect ive wit h l east reci pi ent
and donor si te morbi dit y. Thus i t can be good alternati ve met hod
to bone grafti ng in t he treatment of del ayed uni on of long bones

8. Bone marrow enhances biol ogi cal process of uni on in ri gi dl y
stabilized fractures

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Figure 1b
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Figure 1a
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Table I
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Table II
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Table III
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Table IV
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Table 1
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Table 2
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Table 3
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Table 4
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Table 5
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Table 6
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Table 7
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Table 8
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Table 9
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Table 10
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Table 11
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Table 12
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