WO2008059542A1

WO2008059542A1 - Bone implant with improved surface properties

Info

Publication number: WO2008059542A1
Application number: PCT/IT2006/000805
Authority: WO
Inventors: Clara Cassinelli; Marco Morra; Giovanna Cascardo; Giovanni Agliati; Ignazio Agliati
Original assignee: Nobil Bio Ricerche S.R.L.; Agliati S.R.L.
Priority date: 2006-11-17
Filing date: 2006-11-17
Publication date: 2008-05-22

Abstract

The present invention relates to a bone implant coated with pectin and/or pectin fractions obtainable by treating pectin and/or pectin containing plant tissues with one or more enzymes selected from the group consisting of : cellulase, hemi-cellulase, pectinase (Pgase), pectin-lyase (Pg lyase), pectin methyli esterase, polygalacturonase, acetyl esterase, rhamnogalacturonan hydrolase and lyase, galactanase, arabinanase and xylogalacturonan hydrolase.

Description

DESCRIPTION "Bone implant with improved surface properties"

[0001] The present invention relates to a bone implant, in particular for dental, orthopaedic and spinal surgery prosthetics, with improved surface properties with respect to the devices of the prior art.

[0002] Background of the invention

[0003] The use of metal devices that can be permanently implanted into bone tissue is widespread in various branches of medicine. For example, in the field of dental implant surgery the implantation of screws, generally v made from titanium, into mandibular or maxillary bone tissue, in order to artificially replace lost or dysfunctional bone roots is commonly performed. In the field of orthopaedics, various devices for fixing fractures, for reducing vertebral mobility, for spinal surgery, are commonly implanted into bone tissue.

[0004] In such applications, the implanted device is securely fixed in the implant site thanks to the growth of newly formed bone tissue coming into direct contact with the device itself. This phenomenon, known as osteointegration, has been comprehensively studied and

^■described in the technical-scientific literature relating to this sector, in particular in relation to bone implantation surgery using titanium devices. Contrary to other foreign material implantation processes, involving encapsulation into fibrous material or fibrointegration, the growth of bone tissue coming into direct contact with the device provides solid anchorage, which makes the device itself capable of supporting loads and play a structural role .

[0005] While disciplines based on osteointegration currently enjoy wide success and increasing use, there are still a number of problems to be resolved. In particular, it is important to accelerate the osteointegration process as much as possible, thus reducing the intervening time between insertion of the implant and its effective loading. For example, in ^• dentistry, the implant is generally not "loaded" , and thus the patient cannot exploit the implant for chewing for a period of time which can vary between 1 and 4 months after the operation, so as to allow the bone tissue the chance to heal and induce osteointegration. Furthermore, while the bone healing process generally proceeds well in healthy, young individuals, it is frequently rather slower in older and osteoporotic patients, who require such operations rather more often, and constitute a significant proportion of the patients requiring implant surgery as a result of traumas or in order to fix vertebral mobility.

[0006] In order to overcome the outstanding drawbacks, the recent scientific literature underlines the potential role of cellular behavioural control phenomena at the interface between the implant and host tissue.

[0007] In particular, the literature for the sector highlights the fundamental _:role played by mesenchymal cells present in the bone marrow as precursors in the generation of new tissue in periimplantation bone repair processes. As described by Jose Minguell in the article: Mesenchymal Stem Cells, published in Experimental Biology and Medicine, Volume 226 (6) :507-520, 2001, mesenchymal cells are normally found in an undifferentiated layer in the bone marrow. At the time of fracture, or as a consequence of surgical trauma, for example while inserting a screw implant, these cells migrate from their normal site and, under a complex series of signals and chemotaxis stimuli, reach the wound site. Once at this site they undergo the so-called differentiation process, i.e. they are transformed from unspecialised progenitor cells into cells specifically tasked with new tissue regeneration. Bone marrow mesenchymal cells can

^■differentiate along several specific lineages, in particular, they can be transformed into adipose tissue, muscle tissue, cartilage tissue and bone tissue cells. The mechanisms presiding over the transformation along any given lineage in vivo are somewhat poorly understood in the current art. The reason whereby certain surfaces promote cell adhesion but not differentiation is also poorly understood, while it is known that if cells do not adhere to a surface, then they do not differentiate. [0008] In in vitro cell culture, undifferentiated cells can be directed along the various lineages mentioned by means of soluble agents dissolved in the culture medium, creating the so-called "differentiating media" , as opposed to "basal medium" which maintains the cells in the undifferentiated state.

[0009] Several recent studies indicate that the nature of the surface may in itself act to induce the differentiation of mesenchymal cells, even if the molecular characteristics controlling such transformations are unknown. For example, an article by Salasznyk and co-workers, published in the Journal of Biomedicine and Biotechnology, 2004:1, 24-34, reports that surfaces covered with the extracellular matrix proteins collagen or vitronectin can direct cells along the osteoblastic lineage, even in the absence of soluble differentiating agents. \ [0010] Clearly, the surface of an implantable device coming into contact with bone, capable of spontaneously directing mesenchymal cells along the osteogenic lineage pathway, would have undoubted advantages : promoting osteogenic differentiation, indeed, they can actively give rise to the phenomenon of contact osteogenesis, as described by Professor Davies in the article: Understanding Periimplant Endosseous Healing, published in the Journal of Dental Education, Volume 67, Number 8, August 2003, with possible ^relapse in the quality and quantity of the osteointegration process. [0011] Despite the previous indications, the practical translation of such concepts still gives significant drawbacks due to the lack of knowledge on molecules capable of inducing the differentiation process, or in relation to their suitability for the specific application. For example, collagen may introduce contamination problems due to it being derived from questionable animal sources (particularly bovine collagen) , or rejection due to the possibility of incompatibility between different species. Besides the considerations related to its animal origin, vitronectin is a protein that is present in low quantities in organisms, and is thus very expensive and has poor stability. The uncovered surfaces of certain biocompatible materials, such as titanium and polypropylene, promote the adhesion and differentiation of mesenchymal cells, but at an unsatisfactory rate. [0012] The problem underlying the present invention therefore is that of providing a bone implant with improved surface properties with respect to the devices of the known art, particularly capable of increasing osteoblastic differentiation, thus ensuring faster healing for implanted patients.

[0013] Said problem is resolved by a bone implant as described in the appended claim 1. [0014] In a first aspect, the present invention relates to a bone implant, particularly for dental, orthopaedic and spinal surgery prosthetics, onto the surface of which is immobilised pectin, or a pectin fraction, obtained by v subjecting pectin to enzymatic digestion. [0015] Pectins were discovered in the 19th century, being initially labelled by the family name "pectic acid" , due to the presence of carboxylic acid groups. As described in many texts on the topic, and particularly in the thesis "Studies on the intra- and intermolecular distributions of substituents in commercial pectins" , by Stephanie Guillotin, discussed at the University of Wageningen, in 2005, said molecules have different functional properties and are nowadays commonly used in the food industry as gelling¹, emulsifying and thickening agents . [0016] Figure 1 shows the typical structure of pectins. [0017] Pectins are very complex polysaccharides which can be generally subdivided into three different domains: a domain defined as "smooth" , predominantly composed of polygalacturonan, with very few or no side chains, and two domains defined as "hairy" , which are based on polysaccharides such as Rhamnogalacturonan I and Rhamnogalacturonan II, and which are highly branched. [0018] The various pectin domains can be purified or, in any case, modified, by chemical action (hydrolysis, saponification) or by means of the action of enzymes and enzyme preparations, as described in the literature for the sector. Both pectins and the enzyme preparations required for the degradation thereof, are widely available commercially.

[0019] As will become evident from the experimental tests reported hereinafter, it has surprisingly been discovered, unexpectedly from the data reported in the literature and from the general knowledge of the sector, that pectin or fractions thereof, obtained by means of enzymatic digestion, when bound to the surface of solid substrates, are capable of stimulating the differentiation of bone marrow mesenchymal cells along the osteogenic lineage pathway in a manner superior to that observed with uncoated substrates. [0020] In practice, and in an entirely unexpected way- considering the current state of knowledge, it has been observed that coating materials commonly used in bone implants, particularly titanium, with pectin or certain pectin fractions obtained by means of enzymatic digestion of pectins, results in significant osteogenic stimulation of bone marrow mesenchymal cells, making the material itself more suited to the phenomena of periimplantation healing, according to the lineages indicated, for example in the article cited by Professor Davies. In other words, the layer of pectin or pectin fractions acts as an osteointegration promoting agent, significantly reducing the intervening time between the insertion of the implant into the bone and the restoration of full functionality by the patient. Furthermore, said polysaccharides, being of plant origin, have no tissue contamination problems and are easy to obtain and at limited cost . [0021] Pectins are extracted from the cell walls of fruit, vegetables, or the by-products thereof, that are rich in cell walls, for example the peel. Examples of fruit and vegetables include apple, pear, onion, carrot, leek and potato. Pectins are extracted using the extraction and purification methods known in the sector and which are thus not reported at this time. Alternatively, pectins may be purchased. >■ [0022] The pectin fractions used for the purposes of the invention are obtained by subjecting the pectins, fruit, vegetables, or the pectin-containing by-products thereof to enzymatic digestion according to the methods known in the sector and described, for example, in Schols, efc al.; J. Carbohydr. Res. 1990, 206, 117-129.

[0023] The pectins or fruit, vegetables or the pectin- containing by-products thereof are mixed with water and the pH adjusted to 4-6, preferably at pH of around 5, using a base, for example sodium hydroxide. One or more enzymes, advantageously selected from the group consisting of cellulase, hemi-cellulase, pectinase

(Pgase) , pectin-lyase (Pg lyase) , pectin methyl esterase, polygalacturonase, acetyl esterase, rhamnogalacturonan hydrolase and lyase, galactanase, arabinanase, xylogalacturonan hydrolase and mixtures thereof are added, in quantities of between 0.025 and 0.25% by weight, preferably between 0.05 and 0.15% by weight (the percentage by weight of enzyme is in reference to the quantity of starting product, i.e. pectins, fruit, vegetables or the pectin-containing by-products thereof) . For example, the commercial enzyme preparations Rapidase C600 and Rapidase Liq+ may be used. [0024] The mixture is left incubating for 2-4 hours, preferably for approx. 3 hours, at a temperature of between 40 and 50⁰C, preferably approx. 45⁰C and with constant stirring. Then, a second aliquot of one or more enzymes selected from the above-indicated group, is added in quantities of between 0.025 and 0.25% by weight, preferably between 0.05 and 0.15% by weight, and the incubation continued for 1-2 hours, preferably approx. 1.5 hours. The enzyme is then inactivated by heating the mixture to approx. 90⁰C for approx. half an hour. The pectin fraction thus obtained is finally purified by high pressure size exclusion chromatography (HPSEC) and lyophilised.

[0025] The pectin fraction that can be obtained by means of the above described process essentially comprises the pectin "hairy" regions (HRs) , in that the enzymes mainly degrade the smooth regions,^ i.e. the homogalacturonan domain, thus leaving the HRs.

[0026] Preferably, said hairy regions (HRs) obtained by enzymatic digestion of pectins, are modified hairy regions (MHRs) . Indeed, besides digesting pectin into its "hairy" components, the enzymes, in part, modify the composition and quantity of .. the sugar residues constituting the "hairy" regions, thus giving rise to modified "hairy" regions (MHRs) . [0027] The modified "hairy" regions (MHRs) comprise between 5 to 30 molar percent rhamnose, preferably 10-20 molar percent; 2 to 60 molar percent arabinose, preferably 5-25 molar percent; 7 to 30 molar percent galactose, preferably 10-25 molar percent; 0 to 35 molar percent xylose, preferably 0-25 molar percent; 5 to 60 molar percent galacturonic acid; preferably 10-50 molar percent; 0 to 60 moles of methyl esters per 100 moles of galacturonic acid, preferably 0-50 moles/100 moles of galacturonic acid; 0 to 80 moles of acetyl esters per 100 moles of galacturonic acid, preferably 0-70 moles/100 moles of galacturonic acid. The rhamnose to galacturonic acid ratio is comprised of between 0.10 and 0.60, preferably 0.20 and 0.40.

[0028] Immobilisation of the pectin or pectin fraction comprising HRs and/or MHRs onto the surface of the bone implant, preferably consisting of a biocompatible metal, for example titanium, occurs by means of prior modification of the surface of the implant by means of plasma deposition or polyimine absorption. [0029] With plasma treatment, the surface of the bone implant is functionalised by means of the introduction of amine groups by means of plasma deposition of allylamine. Upon completion of treatment, the implant is immersed in an aqueous solution of pectin or pectin fraction, at a concentration of between 0.1% and 1%, using a suitable coupling agent in order to promote the formation of covalent bonds between the functionalised surface and the polysaccharide. Examples of coupling agents include: carbodiimmide and N-hydroxysuccinimide . The implant is left immersed in the polysaccharide solution for approx. 2-24 hours and then washed with distilled water and air dried. »^■

[0030] In the case of treatment by means of polyimine absorption, the surface of the implant is functionalised with amine groups, by immersing it in a 0.2% aqueous solution of polyimine, preferably polyethylenimine, for 1-3 hours.

[0031] Afterwards, the implant is immersed in an aqueous solution of pectin or pectin fraction under the same conditions as described above, in order to give a bone implant coated with an agent capable of stimulating osteoblastic differentiation,, thus enhancing the rate of bone implant osteointegration. Thus, the present invention allows greatly reduced post-operative healing times with respect to the use of uncoated implants, all advantageous with respect to the health and quality of life of the patients.

[0032] Preferably, prior to functionalisation of the device by means of plasma deposition or polyimine adsorption, the topography of the device metal or polymer surface is altered by performing a roughening or "texturisation" treatment. Such treatments are known in the sector and therefore will not be described here in detail. For example, roughening may be obtained by sanding, plasma spray deposition, acid treatment and electrochemical treatment .

[0033] The bone implants of the invention include, for example, dental prosthetics, such as implantable titanium screws, implantable blades, orthopaedic prosthetics, such as femoral prosthetics and bone prosthetics in general, plates, fixing screws, spinal prosthetics, such as spinal cages, screws, spinal fusion and fixing devices, artificial intervertebral discs and the parts thereof. [0034] The bone implants of the invention are consisting of any biocompatible material suitable for insertion into the human body, for example titanium, steel or biodegradable polymers.

[0035] In one additional aspect, the invention relates to the use of pectin or pectin fractions, for the preparation of a medicament for increasing osteoblastic differentiation. In particular, said polysaccharides increase mesenchymal cell differentiation. The pectin fractions correspond to those'- described above. [0036] EXPERIMENTAL SECTION [0037] Example 1 Attainment of pectin fractions by means of enzymatic treatment

[0038] A certain quantity of apples are homogenised using a blender. The preparation is split into two parts and treated with the following commercial preparations: A) Rapidase C600, manufactured by DSM Food Specialities B) Rapidase Liq+, manufactured by DSM Food Specialities The juice thus obtained is subjected to ultrafiltration and the retentate lyophilised in order to obtain the modified hairy parts (MHR) . [0039] Example 2

Attainment of pectin fractions by means of enzymatic treatment

[0040] 10 kg of apples are homogenised using a blender. 8 kg of the pulp obtained are transferred into an incubation chamber (Terlet, Zutphen, Holland) and mixed with 3 litres of water. The pH is adjusted to 5 by means of the addition of 5 M NaOH, and 10 g of enzyme added

(Rapidase Liq+) . Incubation is carried out at 45⁰C with constant stirring. After 3 hours, 5 g of enzyme are added and the incubation continued for 1.5 hours (4.5 hours total) . The enzyme is inactivated by heating the solution to 90⁰C for 30 minutes. The modified "hairy" regions are isolated and purified by means of high pressure size exclusion chromatography (HPSEC) , using three TosoHaas TSK-GeI G columns (4000PWXL-3000PWXL-2500PWXL) in series and 0.2 M sodium nitrate as eluent [0041] Composition of the MHRs

sugar MHR

(molar%)

Rhamnose 11

Arabinose 11

Galactose 20

Glucose 3

Xylose 18

Galacturonic acid 37

Methyl esters/100 mol of 34 galacturonic acid

Acetyl esters/100 mol of 11 galacturonic acid

Rhamnose :Galacturonic acid 0.30

[0042] Example 3 \

[0043] Binding MHRs to the surface of solids

[0044] Three 1 cm squares of 99.7% titanium (Sigma- Aldrich) , are subjected to the allylamine plasma deposition process using a Gambetti Kenologia plasma treatment reactor. In particular, the deposition process is carried out by means of 10 ms pulsed plasma cycles, at a pressure of 100 mTorr. The discharge power is 50 W, and the treatment time is 30 s. Upon completion of treatment, the samples are immersed in a solution of MHR prepared as per the previous example, at a concentration of 0.5% in water, using carbodiimide and hydroxysuccinimide (1%) to promote the binding reaction. [0045] Example 4 [0046] Binding MHRs to the surface of solids [0047] The reaction in the previous example is carried out as reported above. However, the surface aminisation step is not achieved by means of plasma deposition, but by means of the absorption of polyethyleneimine (PEI, Aldrich) from a 0.2% solution in water, for 2 hours.

[0048] Example 5 v

[0049] Evaluation of MHR coated surface induced mesenchymal cell differentiation. [0050] The reaction described in example 3 is repeated on polystyrene cell culture plates, thus giving MHR coated polystyrene. A further set of samples is produced by covering the polystyrene with whole commercial pectin, with high methoxy content (High Methoxy (HM) Pectin) , while uncoated cell culture polystyrene is used as a control .

[0051] The cells used for the test are normal human bone marrow mesenchymal cells ^l (hMSC) purchased from the Cambrex cell culture bank through Cambrex Bio Science Italia S.r.l. The purity of the cell lineage and the capacity to differentiate along the osteogenic, chondrogenic and adipogenic pathways, have been verified by means of flow cytometry. Tests have been conducted according to the protocols listed in the international bibliography. In particular, half samples have been subjected to evaluation of alkaline phosphatase (ALP) activity, in both osteogenic and non-osteogenic media. The other half samples have been used for the cell proliferation tests, evaluated by means of the MTT test,

5 again in this case, both in osteogenic and non-osteogenic media. The MTT test measures the efficiency of the mitochondrial Krebs cycle enzyme succinate dehydrogenase

(SDH) , an oxidoreductase which catalyses the conversion of succinic acid to fumaric acid. Since this conversion

10 only occurs in living cells, it is universally accepted as an effective method for measuring cell proliferation and, in the case of reduced enzyme activity, the presence of toxic effects. i

[0052] A suspension of 4.33 x 10⁴ + 1.25 x 10⁴ hMSC cells

15 (obtained by means of the addition of 2 mL of trypsin/EDTA solution to the monolayer contained in a T75

Falcon flask) in 2 mL of hMSC Differentiation BulletKit-

Osteogenic medium (Biowhittaker, Cambrex Bio Science

Italia S.r.l., Milan) is introduced into a sterile

20. polystyrene container. At the same time, a suspension with the same number of cells, but in 2 mL of Mesenchymal

Stem Cell Growth Medium BulletKit (Biowhittaker, Cambrex

Bio Science Italia S.r.l., Milan), was introduced into another sterile polystyrene container. Each container

25 holds 6 samples of polystyrene modified with MHR, 6 samples of unmodified polystyrene and one control, into which is added just culture medium without cells. The containers are subsequently placed in an incubator at 37⁰C, 5% CO₂ and relative humidity of 98%, some for 3 days, others for 7, and others for 10. At the end of each growth period, 3 samples were used for the evaluation of alkaline phosphatase activity, 3 for the evaluation of cell proliferation by means of the MTT test. [0053] For the evaluation of ALP, after washing in DPBS, 0.5 mL of a solution of 0.1% Triton X-1001 (Sigma Aldrich S.r.l.) was added and the suspension maintained for 30 minutes in an incubator at 37⁰C so as to permit lysis of the cells growing on the surface. Subsequently, to 0.5 mL of each cell lysate was added 0.5 mL of reaction mixture, composed of an equal volume of 2 mM MgCl₂.7H₂O, 3 mM p-

Nitrophenylphosphate and 2 mM 2-amino-2-methyl-1-propanol

(AMP) . After incubation for 30 minutes at 37⁰C, the reaction was terminated using 1 N NaOH. The absorbance at

405 nm was read using a GENIUS TECAN plate reader. The enzyme alkaline phosphatase (ALP) catalyses the transformation of p-nitrophenylphosphate into paranitrophenol according to the reported scheme: ALP p-nitrophenylphosphate > p-nitrophenol + H₃PO₄. (colourless) 405 nm (yellow) 5

19

[0054] The value for ALP activity was normalised for the value of the absorbance produced by the samples of the same family subjected to the MTT test and the phosphatase activity expressed as the specific enzyme activity.

For the MTT test, after washing in phosphate buffer, a solution of 5 mg/mL of a soluble tetrazolium salt (in particular, 3- (4, 5-dimethylthiazol-2yl) -2 , 5 diphenyl tetrazolium bromide) was added. During the following three hours of incubation at 37°C, the enzyme succinate dehydrogenase induces the transformation of the tetrazolium salts, initially soluble and yellow in colour, into a blue-coloured water insoluble product, the formazan: the greater the quantity of precipitate, the higher the level of enzyme activity and, consequently, the number of metabolically viable cells. The precipitate was solubilised with acidic isopropanol (2% IN HCl) and measured spectrophotometrically at a wavelength of 560 nm by means of a Genios plate reader (TECAN S . r.1. ) . [0055] The results obtained show that, in osteogenic medium, the value recorded for the MHR coated polystyrene

(PSMHR) is significantly different from that recorded on cell culture polystyrene and from that of the control without cells. Cell adhesioή. occurs on both cell culture polystyrene and on MHR coated polystyrene, but the PSMHR promotes substantially enhanced osteoblastic differentiation with respect to uncoated polystyrene.

[0056] Even in basal medium, i.e. in the absence of soluble stimuli for inducing differentiation, the PSMHR induces significantly higher alkaline phosphatase expression with respect to uncoated polystyrene, thus confirming the unexpected effect on osteogenic differentiation.

[0057] Example 6

[0058] Evaluation of the _:superiority in promoting mesenchymal cell differentiation by MHR coated Titanium with respect to Titanium alone.

[0059] Commercially pure Titanium (cpTi) disks are prepared as follows :

[0060] 1) no treatment (Smooth Ti) ; [0061] 2) acid double etching treatment according to that envisaged for achieving an Osseotite roughened surface (3i) ;

[0062] 3) Smooth Ti onto which is bound MHR, as in example

2; [0063] 4) control comprising just culture medium without cells.

[0064] The samples prepared are used for evaluating osteoblastic differentiation by means of measuring specific alkaline phosphatase activity, as for the previous example. The tests are performed in differentiating medium. The following results are obtained (the numbers report the absorbance measured using a Tecan microplate reader. .Obviously, the higher the number, the greater the enzyme activity:

[0065] As can be deduced from the results, after the initial induction period, the cells grown on PSMHR express much higher levels of alkaline phosphatase with respect to titanium, both whether smooth, or with the topography bestowed by the acid etching treatment . The above data indicate that the^ MHR coating significantly, and unexpectedly, improves the ability of the surface to stimulate osteoblastic differentiation, making it significantly better with respect to that provided by the surface of the current material of choice for devices coming into contact with bone .

Claims

v 22Claims

1. A bone implant coated with pectin and/or pectin fractions obtainable by treating pectin and/or pectin containing plant tissues with one or more enzymes selected from the group consisting of: cellulase, hemi-cellulase, pectinase (Pgase) , pectin-lyase (Pg lyase) , pectin methyl esterase, polygalacturonase, acetyl esterase, rhamnogalacturonan hydrolase and lyase, galactanase, arabinanase, xylogalacturonan hydrolase and mixtures thereof.

2. The bone implant according to claim 1 wherein said pectin and/or pectin fractions are immobilised on the surface thereof.

3. The implant according to claims 1 or 2 wherein said pectin-containing plant tissue includes fruit, vegetables or the cell wall-rich by-products thereof .

4. The bone implant according to any of the claims 1 to 3 wherein said treatment is conducted by incubating an aqueous mixture of pectin and/or pectin-containing plant tissues with said enzyme at a temperature of 40-50⁰C for 2-4 hours.

5. The bone implant according to any of the claims 1 to 4 wherein said enzyme is used at a concentration 0805

23 of between 0.025 and 0.25% by weight, preferably between 0.05 and 0.15% by weight.

6. The bone implant according to claims 4 or 5 wherein said mixture of pectin and/or pectin-containing plant tissues and said-, enzyme are incubated for a further 1-2 hours with the prior addition of a second aliquot of enzyme in quantities between 0.025 and 0.25% by weight, preferably between 0.05 and 0.15% by weight.

7. The bone implant according to claim 6 wherein the enzyme is subsequently inactivated by heating said mixture to approx. 90⁰C for approx. half an hour.

8. The bone implant according to claim 7 wherein the pectin fractions are subsequently purified by means of high pressure size exclusion chromatography (HPSEC) and then lyophilised.

9. The bone implant according to any of the claims 1 to 8 wherein said pectin fractions comprise pectin "hairy" regions (HRs) and/or modified "hairy" regions (MHRs) , said modified "hairy" regions comprising: 5 to 30 molar percent rhamnose, 2-60 molar percent arabinose, 7-30 molar percent galactose, 0-35 molar percent xylose, 5-60 molar percent galacturonic acid; 0-60 moles of methyl esters per 100 moles of galacturonic acid, 0-80 v moles of acetyl esters per 100 moles of galacturonic acid.

10. The bone implant according to claim 9 wherein said modified "hairy" regions comprise: 10 to 20 molar percent rhamnose; 5-25 molar percent arabinose; 10-

25 molar percent galactose; 0-25 molar percent xylose; 10-50 molar percent galacturonic acid; 0-50 moles of methyl esters per 100 moles of galacturonic acid; 0-70 moles of acetyl esters per 100 moles of galacturonic acid.

11. The bone implant according to claims 9 or 10 wherein the ratio between rhamnose and galacturonic acid is between 0.10 and 0.60, preferably 0.20 and

0.40.

12. The bone implant according to any of the claims 1 to 11 wherein said implant is made of metal or a polymer.

13. The bone implant according to claim 12 wherein said implant is made of titanium, steel or a biodegradable polymer.

14. The bone implant according to any of the claims 1 to 13 wherein said implant is a dental, orthopaedic or spinal prosthetic.

15. The bone implant according to claim 14 wherein said dental prosthetics include: implantable titanium screws, implantable blades; said orthopaedic prosthetics include: femoral prosthetics and bone prosthetics in general, plates, fixing screws; said spinal prosthetics include: spinal cages, screws, spinal fusion and fixing devices, artificial intervertebral discs and^ the parts thereof.

16. A process for manufacturing a bone implant according to any of the claims 1 to 15 comprising the steps of : a) Functionalising the surface of the implant by- means of plasma deposition or polyimine absorption. b) immobilising said pectins and/or pectin fractions on the functionalised surface of said implant .

17. The process according to claim 16 wherein, in the case of plasma deposition, the surface of the implant is functionalised by means of the introduction of amine groups through the plasma deposition of allylamine.

18. The process according to claims 16 or 17 wherein said functionalised implant is immersed in an aqueous solution of pectin and/or pectin fractions for 2-24 hours in the presence of a coupling agent.

19. The process according to claim 16 wherein, in the case of polyimine absorption, the surface of said implant is functionalised with amine groups by immersing it in an aqueous solution of polyimine for 1-3 hours.

20. The process according to ciaims 16 or 19 wherein said functionalised implant is immersed in a solution of pectin and/or pectin fractions for 2-24 hours in the presence of a coupling agent .

21. The process according to any of the claims 16 to 20 comprising, prior to step a) , a further step a₀) for modifying the surface topography of the implant .

22. The process according to claim 21 wherein said step a₀) comprises a roughening or "texturisation" treatment .

23. The process according to claim 22 wherein said roughening is carried out by sanding, plasma spray deposition, acid treatment and electrochemical treatment .

24. Use of pectin and/or pectin fractions obtainable by treating pectin and/or pectin containing plant tissues with one or more enzymes selected from the group consisting of : cellulase, hemi-cellulase, pectinase (Pgase) , pectin-lyase (Pg lyase) , pectin methyl esterase, polygalacturonase, acetyl esterase, rhaπmogalacturonan hydrolase and lyase, galactanase, arabinanase, xylogalacturonan hydrolase and mixtures thereof, for the preparation of a medicament for stimulating osteoblastic differentiation.

25. The use according to claim 24 for stimulating mesenchymal cell differentiation.

26. The use according to claims 24 or 25 wherein said pectin fractions comprise pectin "hairy" regions (HRs) and/or modified "hairy" regions (MHRs) , said modified "hairy" regions comprising: 5 to 30 molar percent rhamnose, 2-60 molar percent arabinose, 7- 30 molar percent galactose, 0-35 molar percent xylose, 5-60 molar percent galacturonic acid; 0-60 moles of methyl esters per 100 moles of galacturonic acid, 0-80 moles of acetyl esters per 100 moles of galacturonic acid.

27. The use according to claim 26 wherein said modified "hairy" regions comprise: 10 to 20 molar percent rhamnose; 5-25 molar percent arabinose; 10-25 molar percent galactose; 0-25 molar percent xylose; 10-50 molar percent galacturonic acid; 0-50 moles of methyl esters per 100 moles of galacturonic acid; 0-70 moles of acetyl esters per 100 moles of galacturonic acid.

28. The use according to claims 26 or 27 wherein the ratio between rhamnose¹ and galacturonic acid is between 0.10 and 0.60, preferably 0.20 and 0.40.